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CONTENTS i
FOURTEENTH EDITION
Junqueira’s
Basic Histology T E X T A N D AT L A S
Anthony L. Mescher, PhD

Professor of Anatomy and Cell Biology
Indiana University School of Medicine
Bloomington, Indiana
New York Chicago San Francisco Athens London Madrid Mexico City
Milan New Delhi Singapore Sydney Toronto
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Contents
KEY FEATURESâ•‡ VIâ•‡ |â•‡ PREFACEâ•‡IXâ•‡|â•‡ACKNOWLEDGMENTSâ•‡XI
1 Histology & Its Methods 5 Connective Tissueâ•‡ 96

of Studyâ•‡ 1 Cells of Connective Tissueâ•‡ 96
Preparation of Tissues for Studyâ•‡ 1 Fibersâ•‡103
Light Microscopyâ•‡ 4 Ground Substanceâ•‡ 111
Electron Microscopyâ•‡ 8 Types of Connective Tissueâ•‡ 114
Autoradiographyâ•‡9 Summary of Key Pointsâ•‡ 119
Cell & Tissue Cultureâ•‡ 10 Assess Your Knowledgeâ•‡ 120
Enzyme Histochemistryâ•‡ 10
Visualizing Specific Moleculesâ•‡ 10 6 Adipose Tissueâ•‡ 122
Interpretation of Structures in Tissue White Adipose Tissueâ•‡ 122
Sectionsâ•‡14 Brown Adipose Tissueâ•‡ 126
Summary of Key Pointsâ•‡ 15 Summary of Key Pointsâ•‡ 127
Assess Your Knowledgeâ•‡ 16 Assess Your Knowledgeâ•‡ 128
2 The Cytoplasmâ•‡ 17 7 Cartilageâ•‡129

Cell Differentiationâ•‡ 17 Hyaline Cartilageâ•‡ 129
The Plasma Membraneâ•‡ 17 Elastic Cartilageâ•‡ 133
Cytoplasmic Organellesâ•‡ 27 Fibrocartilageâ•‡134
The Cytoskeletonâ•‡ 42 Cartilage Formation, Growth, & Repairâ•‡ 134
Inclusionsâ•‡47 Summary of Key Pointsâ•‡ 136
Summary of Key Pointsâ•‡ 51 Assess Your Knowledgeâ•‡ 136
Assess Your Knowledgeâ•‡ 52
8 Boneâ•‡138
3 The Nucleusâ•‡ 53 Bone Cellsâ•‡ 138
Components of the Nucleusâ•‡ 53 Bone Matrixâ•‡ 143
The Cell Cycleâ•‡ 58 Periosteum & Endosteumâ•‡ 143
Mitosisâ•‡61 Types of Boneâ•‡ 143
Stem Cells & Tissue Renewalâ•‡ 65 Osteogenesisâ•‡148
Meiosisâ•‡65 Bone Remodeling & Repairâ•‡ 152
Apoptosisâ•‡67 Metabolic Role of Boneâ•‡ 153
Summary of Key Pointsâ•‡ 69 Jointsâ•‡155
Assess Your Knowledgeâ•‡ 70 Summary of Key Pointsâ•‡ 158
4 Epithelial Tissueâ•‡ 71
Characteristic Features of Epithelial Cellsâ•‡ 72 9 Nerve Tissue & the Nervous
Specializations of the Apical Cell Surfaceâ•‡ 77 Systemâ•‡161
Types of Epitheliaâ•‡ 80 Development of Nerve Tissueâ•‡ 161
Transport Across Epitheliaâ•‡ 88 Neuronsâ•‡163
Renewal of Epithelial Cellsâ•‡ 88 Glial Cells & Neuronal Activityâ•‡ 168
Summary of Key Pointsâ•‡ 90 Central Nervous Systemâ•‡ 175
Assess Your Knowledgeâ•‡ 93 Peripheral Nervous Systemâ•‡ 182
iii
iv CONTENTS
Neural Plasticity & Regenerationâ•‡ 187 15 Digestive Tractâ•‡ 295

Summary of Key Pointsâ•‡ 190
General Structure of the Digestive Tractâ•‡ 295
Oral Cavityâ•‡ 298
Esophagusâ•‡305
10 Muscle Tissueâ•‡ 193 Stomachâ•‡307
Skeletal Muscleâ•‡ 193 Small Intestineâ•‡ 314
Cardiac Muscleâ•‡ 207 Large Intestineâ•‡ 318
Smooth Muscleâ•‡ 208 Summary of Key Pointsâ•‡ 326
Regeneration of Muscle Tissueâ•‡ 213 Assess Your Knowledgeâ•‡ 328
Assess Your Knowledgeâ•‡ 214 16 Organs Associated with the Digestive
Tractâ•‡329
11 The Circulatory Systemâ•‡ 215 Salivary Glandsâ•‡ 329
Heartâ•‡215 Pancreasâ•‡332
Tissues of the Vascular Wallâ•‡ 219 Liverâ•‡335
Vasculatureâ•‡220 Biliary Tract & Gallbladderâ•‡ 345
Lymphatic Vascular Systemâ•‡ 231 Summary of Key Pointsâ•‡ 346
17 The Respiratory Systemâ•‡ 349

12 Bloodâ•‡ 237 Nasal Cavitiesâ•‡ 349
Composition of Plasmaâ•‡ 237 Pharynxâ•‡352
Blood Cellsâ•‡ 239 Larynxâ•‡352
Summary of Key Pointsâ•‡ 250 Tracheaâ•‡354
Assess Your Knowledgeâ•‡ 252 Bronchial Tree & Lungâ•‡ 354
Lung Vasculature & Nervesâ•‡ 366
13 Hemopoiesisâ•‡ 254 Pleural Membranesâ•‡ 368
Stem Cells, Growth Factors, & Differentiationâ•‡ 254 Respiratory Movementsâ•‡ 368
Bone Marrowâ•‡ 255 Summary of Key Pointsâ•‡ 369
Maturation of Erythrocytesâ•‡ 258 Assess Your Knowledgeâ•‡ 369
Maturation of Granulocytesâ•‡ 260
Maturation of Agranulocytesâ•‡ 263 18 Skinâ•‡ 371
Origin of Plateletsâ•‡ 263 Epidermisâ•‡372
Summary of Key Pointsâ•‡ 265 Dermisâ•‡378
Assess Your Knowledgeâ•‡ 265 Subcutaneous Tissueâ•‡ 381
Sensory Receptorsâ•‡ 381
14 The Immune System & Lymphoid Hairâ•‡383
Organsâ•‡267 Nailsâ•‡384
Innate & Adaptive Immunityâ•‡ 267 Skin Glandsâ•‡ 385
Cytokinesâ•‡269 Skin Repairâ•‡ 388
Antigens & Antibodiesâ•‡ 270 Summary of Key Pointsâ•‡ 391
Antigen Presentationâ•‡ 271 Assess Your Knowledgeâ•‡ 391
Cells of Adaptive Immunityâ•‡ 273
Thymusâ•‡276 19 The Urinary Systemâ•‡ 393
Mucosa-Associated Lymphoid Tissueâ•‡ 281 Kidneysâ•‡393
Lymph Nodesâ•‡ 282 Blood Circulationâ•‡ 394
Spleenâ•‡286 Renal Function: Filtration, Secretion, &
Summary of Key Pointsâ•‡ 293 Reabsorptionâ•‡395
Assess Your Knowledgeâ•‡ 294 Ureters, Bladder, & Urethraâ•‡ 406
CONTENTS v
Summary of Key Pointsâ•‡ 411 22 The Female Reproductive Systemâ•‡ 460

Ovariesâ•‡460
Uterine Tubesâ•‡ 470
20 Endocrine Glandsâ•‡ 413 Major Events of Fertilizationâ•‡ 471
Pituitary Gland (Hypophysis)â•‡ 413 Uterusâ•‡471
Adrenal Glandsâ•‡ 423 Embryonic Implantation, Decidua, & the Placentaâ•‡ 478
Pancreatic Isletsâ•‡ 427 Cervixâ•‡482
Diffuse Neuroendocrine Systemâ•‡ 429 Vaginaâ•‡483
Thyroid Glandâ•‡ 429 External Genitaliaâ•‡ 483
Parathyroid Glandsâ•‡ 432 Mammary Glandsâ•‡ 483
Pineal Glandâ•‡ 434 Summary of Key Pointsâ•‡ 488
23 The Eye & Ear: Special Sense
21 The Male Reproductive Organsâ•‡490
Systemâ•‡439 Eyes: The Photoreceptor Systemâ•‡ 490
Testesâ•‡439 Ears: The Vestibuloauditory Systemâ•‡ 509
Intratesticular Ductsâ•‡ 449 Summary of Key Pointsâ•‡ 522
Excretory Genital Ductsâ•‡ 450 Assess Your Knowledgeâ•‡ 522
Accessory Glandsâ•‡ 451
Penisâ•‡456 APPENDIXâ•‡525
FIGURE CREDITSâ•‡527
INDEXâ•‡529
Preface
With this 14th edition, Junqueira’s Basic Histology continues as throughout the book as needed, and again make up a complete
the preeminent source of concise yet thorough information atlas of cell, tissue, and organ structures fully compatible
on human tissue structure and function. For nearly 45 years with the students’ own collection of glass or digital slides. A
this educational resource has met the needs of learners for a virtual microscope with over 150 slides of all human tissues
well-organized and concise presentation of cell biology and and organs is available: http://medsci.indiana.edu/junqueira/
histology that integrates the material with that of biochemistry, virtual/junqueira.htm.
immunology, endocrinology, and physiology and provides As with the previous edition, the book facilitates learning
an excellent foundation for subsequent studies in pathology. by its organization:
The text is prepared specifically for students of medicine
and other health-related professions, as well as for advanced
■ An opening chapter reviews the histological techniques
that allow understanding of cell and tissue structure.
undergraduate courses in tissue biology. As a result of its value
and appeal to students and instructors alike, Junqueira’s Basic
■ Two chapters then summarize the structural and
functional organization of human cell biology,
Histology has been translated into a dozen different languages
presenting the cytoplasm and nucleus separately.
and is used by medical students throughout the world.
This edition now includes with each chapter a set of
■ The next seven chapters cover the four basic tissues that
make up our organs: epithelia, connective tissue (and its
multiple-choice Self-Test Questions that allow readers to assess
major sub-types), nervous tissue, and muscle.
their comprehension and knowledge of important material in
that chapter. At least a few questions in each set utilize clinical
■ Remaining chapters explain the organization and
functional significance of these tissues in each of
vignettes or cases to provide context for framing the medical
the body’s organ systems, closing with up-to-date
relevance of concepts in basic science, as recommended by
consideration of cells in the eye and ear.
the US National Board of Medical Examiners. As with the
last edition, each chapter also includes a Summary of Key For additional review of what’s been learned or to
Points designed to guide the students concerning what is assist rapid assimilation of the material in Junqueira’s Basic
clearly important and what is less so. Summary Tables in Histology, McGraw-Hill has published a set of 200 full-color
each chapter organize and condense important information, Basic Histology Flash Cards, Anthony Mescher author. Each
further facilitating efficient learning. card includes images of key structures to identify, a summary
Each chapter has been revised and shortened, while of important facts about those structures, and a clinical
coverage of specific topics has been expanded as needed. Study comment. This valuable learning aid is available as a set of
is facilitated by modern page design. Inserted throughout each actual cards from Amazon.com, or as an app for smart phones
chapter are more numerous, short paragraphs that indicate how or tablets from the online App Store.
the information presented can be used medically and which With its proven strengths and the addition of new features,
emphasize the foundational relevance of the material learned. I am confident that Junqueira’s Basic Histology will continue
The art and other figures are presented in each chapter, as one of the most valuable and most widely read educational
with the goal to simplify learning and integration with resources in histology. Users are invited to provide feedback
related material. The McGraw-Hill medical illustrations, now to the author with regard to any aspect of the book’s features.
used throughout the text and supplemented by numerous
animations in the electronic version of the text, are the Anthony L. Mescher
most useful, thorough, and attractive of any similar medical Indiana University School of Medicine
textbook. Electron and light micrographs have been replaced mescher@indiana.edu
ix
Acknowledgments
I wish to thank the students at Indiana University School of these valuable resources is gratefully acknowledged. Students are
Medicine and the undergraduates at Indiana University with referred to those review books for hundreds of additional self-
whom I have studied histology and cell biology for over 30 years assessment questions.
and from whom I have learned much about presenting basic I am also grateful to my colleagues and reviewers from
concepts most effectively. Their input has greatly helped in the throughout the world who provided specialized expertise
task of maintaining and updating the presentations in this classic or original photographs, which are also acknowledged in
textbook. As with the last edition the help of Sue Childress and figure captions. I thank those professors and students in the
Dr. Mark Braun was invaluable in slide preparation and the United States, as well as Argentina, Canada, Iran, Ireland, Italy,
virtual microscope for human histology respectively. Pakistan, and Syria, who provided useful suggestions that
A major change in this edition is the inclusion of self- have improved the new edition of Junqueira’s Basic Histology.
assessment questions with each topic/chapter. Many of these Finally, I am pleased to acknowledge the help and collegiality
questions were used in my courses, but others are taken or provided by the staff of McGraw-Hill, especially editors
modified from a few of the many excellent review books published Michael Weitz and Brian Kearns, whose work made possible
by McGraw-Hill/Lange for students preparing to take the U.S. publication of this 14th edition of Junqueira’s Basic Histology.
Medical Licensing Examination. These include Histology and Cell
Biology: Examination and Board Review, by Douglas Paulsen; Anthony L. Mescher
USMLE Road Map: Histology, by Harold Sheedlo; and Anatomy,
Indiana University School of Medicine
Histology, & Cell Biology: PreTest Self-Assessment & Review, by
Robert Klein and George Enders. The use here of questions from mescher@indiana.edu
xi
C H A P T E R
1
PREPARATION OF TISSUES FOR STUDY
Histology & Its
Methods of Study
1 AUTORADIOGRAPHY 9
Fixation 1 CELL & TISSUE CULTURE 10
Embedding & Sectioning 3
ENZYME HISTOCHEMISTRY 10
Staining 3
LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10
Bright-Field Microscopy 4 Immunohistochemistry 11
Fluorescence Microscopy 5 Hybridization Techniques 12
Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE
Confocal Microscopy 5 SECTIONS 14
Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15
ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16
Transmission Electron Microscopy 8
Scanning Electron Microscopy 9
H istology is the study of the tissues of the body and

how these tissues are arranged to constitute organs.
This subject involves all aspects of tissue biology, with
the focus on how cells’ structure and arrangement optimize
functions specific to each organ.
a better knowledge of tissue biology. Familiarity with the tools
and methods of any branch of science is essential for a proper
understanding of the subject. This chapter reviews common
methods used to study cells and tissues, focusing on micro-
scopic approaches.
Tissues have two interacting components: cells and
extracellular matrix (ECM). The ECM consists of many kinds
of macromolecules, most of which form complex structures,
such as collagen fibrils. The ECM supports the cells and
›â•ºPREPARATION OF TISSUES
contains the fluid transporting nutrients to the cells, and FOR STUDY
carrying away their wastes and secretory products. Cells The most common procedure used in histologic research is
produce the ECM locally and are in turn strongly influenced the preparation of tissue slices or “sections” that can be exam-
by matrix molecules. Many matrix components bind to spe- ined visually with transmitted light. Because most tissues and
cific cell surface receptors that span the cell membranes and organs are too thick for light to pass through, thin translu-
connect to structural components inside the cells, forming a cent sections are cut from them and placed on glass slides for
continuum in which cells and the ECM function together in microscopic examination of the internal structures.
a well-coordinated manner. The ideal microscopic preparation is preserved so that the
During development, cells and their associated matrix tissue on the slide has the same structural features it had in the
become functionally specialized and give rise to fundamen- body. However, this is often not feasible because the prepara-
tal types of tissues with characteristic structural features. tion process can remove cellular lipid, with slight distortions
Organs are formed by an orderly combination of these tissues, of cell structure. The basic steps used in tissue preparation for
and their precise arrangement allows the functioning of each light microscopy are shown in Figure 1–1.
organ and of the organism as a whole.
The small size of cells and matrix components makes his-
tology dependent on the use of microscopes and molecular Fixation
methods of study. Advances in biochemistry, molecular biol- To preserve tissue structure and prevent degradation by
ogy, physiology, immunology, and pathology are essential for enzymes released from the cells or microorganisms, pieces of
1
2 CHAPTER 1â•… ■â•… Histology & Its Methods of Study
FIGURE 1–1â•‡ Sectioning fixed and embedded tissue.
52°- 60°C
(a) Fixation Dehydration Clearing Infiltration Embedding
Drive wheel
Block holder
Paraffin block
Tissue
Steel knife
Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec-
this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating
solutions are used with smaller tissue samples and embedding
■âŒ€ Fixation: Small pieces of tissue are placed in solutions of
involves epoxy resins which become harder than paraffin to allow
chemicals that cross-link proteins and inactivate degradative
very thin sectioning.
enzymes, which preserves cell and tissue structure.
■âŒ€ Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues
increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted
which removes all water. in the paraffin block holder, and each turn of the drive wheel by
■âŒ€ Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener-
both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block
■âŒ€ Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness
becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections
■âŒ€ Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized,
mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than
■âŒ€ Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an
the tissue for sectioning (slicing) on a microtome. ultramicrotome with a glass or diamond knife.
organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins,
body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral-
called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and
the tissues to preserve all cells, tissues are usually cut into small ECM structures.
fragments before fixation to facilitate penetration. To improve Electron microscopy provides much greater magnification
cell preservation in large organs fixatives are often introduced and resolution of very small cellular structures and fixation
via blood vessels, with vascular perfusion allowing fixation must be done very carefully to preserve additional “ultra-
rapidly throughout the tissues. structural” detail. Typically in such studies glutaraldehyde-
One widely used fixative for light microscopy is forma- treated tissue is then immersed in buffered osmium
lin, a buffered isotonic solution of 37% formaldehyde. Both tetroxide, which preserves (and stains) cellular lipids as well
this compound and glutaraldehyde, a fixative used for electron as proteins.
Preparation of Tissues for Study 3
Embedding & Sectioning Staining
C H A P T E R
To permit thin sectioning fixed tissues are infiltrated and Most cells and extracellular material are completely color-
embedded in a material that imparts a firm consistency. less, and to be studied microscopically tissue sections must
Embedding materials include paraffin, used routinely for light be stained (dyed). Methods of staining have been devised that
microscopy, and plastic resins, which are adapted for both make various tissue components not only conspicuous but also
light and electron microscopy. distinguishable from one another. Dyes stain material more or
Before infiltration with such media the fixed tissue must less selectively, often behaving like acidic or basic compounds
undergo dehydration by having its water extracted gradually and forming electrostatic (salt) linkages with ionizable radicals
1
by transfers through a series of increasing ethanol solutions, of macromolecules in tissues. Cell components such as nucleic
Histology & Its Methods of Study â•‡ ■â•‡ Preparation of Tissues for Study
ending in 100% ethanol. The ethanol is then replaced by an acids with a net negative charge (anionic) have an affinity for
organic solvent miscible with both alcohol and the embedding basic dyes and are termed basophilic; cationic components,
medium, a step referred to as clearing because infiltration with such as proteins with many ionized amino groups, stain more
the reagents used here gives the tissue a translucent appearance. readily with acidic dyes and are termed acidophilic.
The fully cleared tissue is then placed in melted paraffin Examples of basic dyes include toluidine blue, alcian blue,
in an oven at 52°-60°C, which evaporates the clearing solvent and methylene blue. Hematoxylin behaves like a basic dye,
and promotes infiltration of the tissue with paraffin, and then staining basophilic tissue components. The main tissue com-
embedded by allowing it to harden in a small container of ponents that ionize and react with basic dyes do so because of
paraffin at room temperature. Tissues to be embedded with acids in their composition (DNA, RNA, and glycosaminogly-
plastic resin are also dehydrated in ethanol and then infiltrated cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain
with plastic solvents that harden when cross-linking polymer- the acidophilic components of tissues such as mitochondria,
izers are added. Plastic embedding avoids the higher tempera- secretory granules, and collagen.
tures needed with paraffin, which helps avoid tissue distortion. Of all staining methods, the simple combination of
The hardened block with tissue and surrounding embed- hematoxylin and eosin (H&E) is used most commonly.
ding medium is trimmed and placed for sectioning in an Hematoxylin stains DNA in the cell nucleus, RNA-rich por-
instrument called a microtome (Figure 1–1). Paraffin sections tions of the cytoplasm, and the matrix of cartilage, produc-
are typically cut at 3-10 μm thickness for light microscopy, but ing a dark blue or purple color. In contrast, eosin stains other
electron microscopy requires sections less than 1 μm thick. cytoplasmic structures and collagen pink (Figure 1–2a). Here
One micrometer (1 μm) equals 1/1000 of a millimeter (mm) eosin is considered a counterstain, which is usually a single
or 10–6 m. Other spatial units commonly used in microscopy dye applied separately to distinguish additional features of a
are the nanometer (1 nm = 0.001 μm = 10–6 mm = 10–9 m) and tissue. More complex procedures, such as trichrome stains (eg,
angstrom (1 Å = 0.1 nm or 10–4 μm). The sections are placed Masson trichrome), allow greater distinctions among various
on glass slides and stained for light microscopy or on metal extracellular tissue components.
grids for electron microscopic staining and examination. The periodic acid-Schiff (PAS) reaction utilizes the
hexose rings of polysaccharides and other carbohydrate-rich
› â•ºâ•º MEDICAL APPLICATION tissue structures and stains such macromolecules distinctly
purple or magenta. Figure 1–2b shows an example of cells with
Biopsies are tissue samples removed during surgery or rou- carbohydrate-rich areas well-stained by the PAS reaction. The
tine medical procedures. In the operating room, biopsies DNA of cell nuclei can be specifically stained using a modifica-
are fixed in vials of formalin for processing and microscopic tion of the PAS procedure called the Feulgen reaction.
analysis in a pathology laboratory. If results of such analyses Basophilic or PAS-positive material can be further identi-
are required before the medical procedure is completed, for fied by enzyme digestion, pretreatment of a tissue section with
example to know whether a growth is malignant before the an enzyme that specifically digests one substrate. For example,
patient is closed, a much more rapid processing method is pretreatment with ribonuclease will greatly reduce cytoplas-
used. The biopsy is rapidly frozen in liquid nitrogen, preserv- mic basophilia with little overall effect on the nucleus, indicat-
ing cell structures and making the tissue hard and ready for ing the importance of RNA for the cytoplasmic staining.
sectioning. A microtome called a cryostat in a cabinet at Lipid-rich structures of cells are revealed by avoiding the
subfreezing temperature is used to section the block with processing steps that remove lipids, such as treatment with
tissue, and the frozen sections are placed on slides for rapid heat and organic solvents, and staining with lipid-soluble
staining and microscopic examination by a pathologist. dyes such as Sudan black, which can be useful in diagnosis
Freezing of tissues is also effective in histochemical stud- of metabolic diseases that involve intracellular accumulations
ies of very sensitive enzymes or small molecules because of cholesterol, phospholipids, or glycolipids. Less common
freezing, unlike fixation, does not inactivate most enzymes. methods of staining can employ metal impregnation tech-
Finally, because clearing solvents often dissolve cell lipids in niques, typically using solutions of silver salts to visual certain
fixed tissues, frozen sections are also useful when structures ECM fibers and specific cellular elements in nervous tissue.
containing lipids are to be studied histologically. The Appendix lists important staining procedures used for
most of the light micrographs in this book.
FIGURE 1–2â•‡ Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining.
G G
G
L
L
a b
Micrographs of epithelium lining the small intestine, (a) stained lumen, where projecting microvilli have a prominent layer of
with H&E, and (b) stained with the PAS reaction for glycoproteins. glycoproteins at the lumen (L) and in the mucin-rich secretory
With H&E, basophilic cell nuclei are stained purple while cyto- granules of goblet cells. Cell surface glycoproteins and mucin are
plasm stains pink. Cell regions with abundant oligosaccharides PAS-positive because of their high content of oligosaccharides
on glycoproteins, such as the ends of the cells at the lumen (L) and polysaccharides respectively. The PAS-stained tissue was
or the scattered mucus-secreting goblet cells (G), are poorly counterstained with hematoxylin to show the cell nuclei. (a. X400;
stained. With PAS, however, cell staining is most intense at the b. X300)
Slide preparation, from tissue fixation to observation (or ocular lens) further magnifying this image and projecting
with a light microscope, may take from 12 hours to 2½ days, it onto the viewer’s retina or a charge-coupled device (CCD)
depending on the size of the tissue, the embedding medium, highly sensitive to low light levels with a camera and monitor.
and the method of staining. The final step before microscopic The total magnification is obtained by multiplying the magni-
observation is mounting a protective glass coverslip on the fying power of the objective and ocular lenses.
slide with clear adhesive. The critical factor in obtaining a crisp, detailed image
with a light microscope is its resolving power, defined as the
smallest distance between two structures at which they can be
›â•ºLIGHT MICROSCOPY seen as separate objects. The maximal resolving power of the
light microscope is approximately 0.2 μm, which can permit
Conventional bright-field microscopy, as well as more special- clear images magnified 1000-1500 times. Objects smaller or
ized applications like fluorescence, phase-contrast, confocal, thinner than 0.2 μm (such as a single ribosome or cytoplasmic
and polarizing microscopy, are all based on the interaction of microfilament) cannot be distinguished with this instrument.
light with tissue components and are used to reveal and study Likewise, two structures such as mitochondria will be seen as
tissue features. only one object if they are separated by less than 0.2 μm. The
microscope’s resolving power determines the quality of the
Bright-Field Microscopy image, its clarity and richness of detail, and depends mainly on
With the bright-field microscope stained tissue is examined the quality of its objective lens. Magnification is of value only
with ordinary light passing through the preparation. As shown when accompanied by high resolution. Objective lenses pro-
in Figure 1–3, the microscope includes an optical system and viding higher magnification are designed to also have higher
mechanisms to move and focus the specimen. The optical resolving power. The eyepiece lens only enlarges the image
components are the condenser focusing light on the object obtained by the objective and does not improve resolution.
to be studied; the objective lens enlarging and projecting the Virtual microscopy, typically used for study of bright-
image of the object toward the observer; and the eyepiece field microscopic preparations, involves the conversion of a
Light Microscopy 5
FIGURE 1–3â•‡ Components and light path of a Fluorescence Microscopy
C H A P T E R
bright-field microscope. When certain cellular substances are irradiated by light of
a proper wavelength, they emit light with a longer wave-
Eyepiece Interpupillar
adjustment
length—a phenomenon called fluorescence. In fluores-
Binocular
tubes Head cence microscopy, tissue sections are usually irradiated with
ultraviolet (UV) light and the emission is in the visible portion
of the spectrum. The fluorescent substances appear bright on
Stand
a dark background. For fluorescent microscopy the instru-
1
Measuring
ment has a source of UV or other light and filters that select
Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

graticule
Beamsplitter
rays of different wavelengths emitted by the substances to be
Revolving
nosepiece visualized.
Specimen Objective Fluorescent compounds with affinity for specific cell
holder macromolecules may be used as fluorescent stains. Acridine
Mechanical
stage
orange, which binds both DNA and RNA, is an example.
On/off
switch When observed in the fluorescence microscope, these nucleic
Condenser Illumination
intensity
acids emit slightly different fluorescence, allowing them to be
Field lens
control localized separately in cells (Figure 1–4a). Other compounds
Field such as DAPI and Hoechst stain specifically bind DNA and
diaphragm
Collector
are used to stain cell nuclei, emitting a characteristic blue fluo-
lens rescence under UV. Another important application of fluores-
cence microscopy is achieved by coupling compounds such as
X-Y
Base translation fluorescein to molecules that will specifically bind to certain
Tungsten mechanism
cellular components and thus allow the identification of these
halogen
lamp structures under the microscope (Figure 1–4b). Antibodies
labeled with fluorescent compounds are extremely important
Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing
mechanical components and the pathway of light from the Specific Molecules.)
substage lamp to the eye of the observer. The optical system
has three sets of lenses:
■âŒ€ The condenser collects and focuses a cone of light that illu-
Phase-Contrast Microscopy
minates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans-
■âŒ€ Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified
image of the object toward the eyepiece. Interchangeable
light microscopes. Cellular detail is normally difficult to see
objectives with different magnifications routinely used in
histology include X4 for observing a large area (field) of the in unstained tissues because all parts of the specimen have
tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros-
of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images
detailed areas. from transparent objects and, importantly, can be used with
■âŒ€ The two eyepieces or oculars magnify this image another
living, cultured cells (Figure 1–5).
X10 and project it to the viewer, yielding a total magnifica-
tion of X40, X100, or X400. Phase-contrast microscopy is based on the principle
that light changes its speed when passing through cellular
(Used with permission from Nikon Instruments.)
and extracellular structures with different refractive indices.
These changes are used by the phase-contrast system to cause
the structures to appear lighter or darker in relation to each
stained tissue preparation to high-resolution digital images other. Because they allow the examination of cells without
and permits study of tissues using a computer or other digi- fixation or staining, phase-contrast microscopes are promi-
tal device, without an actual stained slide or a microscope. In nent tools in all cell culture laboratories. A modification of
this technique regions of a glass-mounted specimen are cap- phase-contrast microscopy is differential interference
tured digitally in a grid-like pattern at multiple magnifications microscopy with Nomarski optics, which produces an image
using a specialized slide-scanning microscope and saved as of living cells with a more apparent three-dimensional (3D)
thousands of consecutive image files. Software then converts aspect (Figure 1–5c).
this dataset for storage on a server using a format that allows
access, visualization, and navigation of the original slide with
common web browsers or other devices. With advantages in Confocal Microscopy
cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light is rel-
light microscopes and collections of glass slides in histology atively large and fills the specimen. Stray (excess) light reduces
laboratories for students. contrast within the image and compromises the resolving
FIGURE 1–4â•‡ Appearance of cells with fluorescent microscopy.
N R
a b
Components of cells are often stained with compounds visible by filaments show nuclei with blue fluorescence and actin filaments
fluorescence microscopy. stained green. Important information such as the greater density
(a) Acridine orange binds nucleic acids and causes DNA in cell of microfilaments at the cell periphery is readily apparent. (Both
nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to X500)
appear orange in these cells of a kidney tubule. (Figure 1–4b, used with permission from Drs Claire E. Walczak
and Rania Rizk, Indiana University School of Medicine,
(b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) Bloomington.)
that binds DNA and with fluorescein-phalloidin that binds actin
FIGURE 1–5â•‡ Unstained cells’ appearance in three types of light microscopy.
a b c
Living neural crest cells growing in culture appear differently in-phase light differently and produce an image of these features
with various techniques of light microscopy. Here the same field in all the cells.
of unstained cells, including two differentiating pigment cells, is (c) Differential interference microscopy: Cellular details are
shown using three different methods (all X200): highlighted in a different manner using Nomarski optics. Phase-
(a) Bright-field microscopy: Without fixation and staining, only contrast microscopy, with or without differential interference, is
the two pigment cells can be seen. widely used to observe live cells grown in tissue culture.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and (Used with permission from Dr Sherry Rogers, Department of Cell
cytoplasmic structures with different refractive indices affect Biology and Physiology, University of New Mexico, Albuquerque, NM.)
Light Microscopy 7
the specimen allows them to be digitally reconstructed into a

FIGURE 1–6â•‡ Principle of confocal microscopy.
3D image.
C H A P T E R
Laser Polarizing Microscopy
Polarizing microscopy allows the recognition of stained or
unstained structures made of highly organized subunits.
When normal light passes through a polarizing filter, it exits
1
Scanner
vibrating in only one direction. If a second filter is placed in
Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

the microscope above the first one, with its main axis per-
Detector pendicular to the first filter, no light passes through. If, how-
ever, tissue structures containing oriented macromolecules
are located between the two polarizing filters, their repeti-
tive structure rotates the axis of the light emerging from the
polarizer and they appear as bright structures against a dark
background (Figure 1–7). The ability to rotate the direction
of vibration of polarized light is called birefringence and is
Plate with
pinhole
Beam
splitter FIGURE 1–7â•‡ Tissue appearance with bright-field
and polarizing microscopy.
Lens
Other
Focal plane out-of-focus
Specimen planes
Although a very small spot of light originating from one plane

of the section crosses the pinhole and reaches the detector,
rays originating from other planes are blocked by the blind.
Thus, only one very thin plane of the specimen is focused at a a
time. The diagram shows the practical arrangement of a confo-
cal microscope. Light from a laser source hits the specimen and
is reflected. A beam splitter directs the reflected light to a pin-
hole and a detector. Light from components of the specimen
that are above or below the focused plane is blocked by the
blind. The laser scans the specimen so that a larger area of the
specimen can be observed.
power of the objective lens. Confocal microscopy (Figure 1–6)

avoids these problems and achieves high resolution and sharp
focus by using (1) a small point of high-intensity light, often
from a laser, and (2) a plate with a pinhole aperture in front of
the image detector. The point light source, the focal point of b
the lens, and the detector’s pinpoint aperture are all optically
conjugated or aligned to each other in the focal plane (confo-
Polarizing light microscopy produces an image only of material
cal), and unfocused light does not pass through the pinhole. having repetitive, periodic macromolecular structure; features
This greatly improves resolution of the object in focus and without such structure are not seen. Pieces of thin, unsec-
allows the localization of specimen components with much tioned mesentery were stained with red picrosirius, orcein, and
greater precision than with the bright-field microscope. hematoxylin, placed on slides and observed by bright-field (a)
Confocal microscopes include a computer-driven mirror and polarizing (b) microscopy.
system (the beam splitter) to move the point of illumination (a) With bright-field microscopy collagen fibers appear red,
with thin elastic fibers and cell nuclei darker. (X40)
across the specimen automatically and rapidly. Digital images
captured at many individual spots in a very thin plane of focus (b) With polarizing microscopy, only the collagen fibers are vis-
ible and these exhibit intense yellow or orange birefringence.
are used to produce an “optical section” of that plane. Creat- (a: X40; b: X100)
ing such optical sections at a series of focal planes through
a feature of crystalline substances or substances containing The wavelength in an electron beam is much shorter than that
highly oriented molecules, such as cellulose, collagen, micro- of light, allowing a 1000-fold increase in resolution.
tubules, and actin filaments.
The utility of all light microscopic methods is greatly Transmission Electron Microscopy
extended through the use of digital cameras. Many features The transmission electron microscope (TEM) is an imag-
of digitized histological images can be analyzed quantitatively ing system that permits resolution around 3 nm. This high
using appropriate software. Such images can also be enhanced resolution allows isolated particles magnified as much as
to allow objects not directly visible through the eyepieces to be 400,000 times to be viewed in detail. Very thin (40-90 nm),
examined on a monitor. resin-embedded tissue sections are typically studied by TEM
at magnifications up to approximately 120,000 times.
›â•ºELECTRON MICROSCOPY Figure 1–8a indicates the components of a TEM and the
basic principles of its operation: a beam of electrons focused
Transmission and scanning electron microscopes are based on using electromagnetic “lenses” passes through the tissue sec-
the interaction of tissue components with beams of electrons. tion to produce an image with black, white, and intermediate
FIGURE 1–8â•‡ Electron microscopes.
Electron gun Electron gun

Cathode Cathode
3 mm
Anode Anode
Copper grid
Condensor lens with three sections Lens
Specimen Column
Objective lens holder Lens
Column Scanner
Intermediate lens Electron detector
TEM image Lens SEM image

Projector lens
Image on viewing
screen Specimen
Electron detector
with CCD camera
(a) Transmission electron microscope (b) Scanning electron microscope
Electron microscopes are large instruments generally housed in a In a TEM image areas of the specimen through which electrons
specialized EM facility. passed appear bright (electron lucent), while denser areas or
(a) Schematic view of the major components of a transmission elec- those that bind heavy metal ions during specimen preparation
tron microscope (TEM), which is configured rather like an upside- absorb or deflect electrons and appear darker (electron dense).
down light microscope. With the microscope column in a vacuum, a Such images are therefore always black, white, and shades of gray.
metallic (usually tungsten) filament (cathode) at the top emits elec- (b) The scanning electron microscope (SEM) has many similarities
trons that travel to an anode with an accelerating voltage between to a TEM. However, here the focused electron beam does not pass
60 and 120 kV. Electrons passing through a hole in the anode form a through the specimen, but rather is moved sequentially (scanned)
beam that is focused electromagnetically by circular electric coils from point to point across its surface similar to the way an electron
in a manner analogous to the effect of optical lenses on light. beam is scanned across a television tube or screen. For SEM speci-
The first lens is a condenser focusing the beam on the sec- mens are coated with metal atoms with which the electron beam
tion. Some electrons interact with atoms in the section, being interacts, producing reflected electrons and newly emitted secondary
absorbed or scattered to different extents, while others are simply electrons. All of these are captured by a detector and transmitted to
transmitted through the specimen with no interaction. Electrons amplifiers and processed to produce a black-and-white image on the
reaching the objective lens form an image that is then magnified monitor. The SEM shows only surface views of the coated specimen
and finally projected on a fluorescent screen or a charge-coupled but with a striking 3D, shadowed quality. The inside of organs or cells
device (CCD) monitor and camera. can be analyzed after sectioning to expose their internal surfaces.
Autoradiography 9
shades of gray regions. These regions of an electron micro- layer of heavy metal (often gold) which reflects electrons in
graph correspond to tissue areas through which electrons a beam scanning the specimen. The reflected electrons are
C H A P T E R
passed readily (appearing brighter or electron-lucent) and captured by a detector, producing signals that are processed
areas where electrons were absorbed or deflected (appearing to produce a black-and-white image. SEM images are usually
darker or more electron-dense). To improve contrast and reso- easy to interpret because they present a three-dimensional
lution in TEM, compounds with heavy metal ions are often view that appears to be illuminated in the same way that large
added to the fixative or dehydrating solutions used for tissue objects are seen with highlights and shadows caused by light.
preparation. These include osmium tetroxide, lead citrate,
1
and uranyl compounds, which bind cellular macromolecules,
›â•ºAUTORADIOGRAPHY
Histology & Its Methods of Study â•‡ ■â•‡Autoradiography

increasing their electron density and visibility.
Cryofracture and freeze etching are techniques that
Microscopic autoradiography is a method of localizing
allow TEM study of cells without fixation or embedding and
newly synthesized macromolecules in cells or tissue sections.
have been particularly useful in the study of membrane struc-
Radioactively labeled metabolites (nucleotides, amino acids,
ture. In these methods very small tissue specimens are rap-
sugars) provided to the living cells are incorporated into spe-
idly frozen in liquid nitrogen and then cut or fractured with a
cific macromolecules (DNA, RNA, protein, glycoproteins, and
knife. A replica of the frozen exposed surface is produced in a
polysaccharides) and emit weak radiation that is restricted
vacuum by applying thin coats of vaporized platinum or other
to those regions where the molecules are located. Slides with
metal atoms. After removal of the organic material, the replica
radiolabeled cells or tissue sections are coated in a darkroom
of the cut surface can be examined by TEM. With membranes
with photographic emulsion in which silver bromide crystals
the random fracture planes often split the lipid bilayers, expos-
ing protein components whose size, shape, and distribution act as microdetectors of the radiation in the same way that
are difficult to study by other methods. they respond to light in photographic film. After an adequate
exposure time in lightproof boxes, the slides are developed
photographically. Silver bromide crystals reduced by the radia-
Scanning Electron Microscopy tion produce small black grains of metallic silver, which under
Scanning electron microscopy (SEM) provides a high- either the light microscope or TEM indicate the locations of
resolution view of the surfaces of cells, tissues, and organs. Like radiolabeled macromolecules in the tissue (Figure 1–9).
the TEM, this microscope produces and focuses a very narrow Much histological information becomes available by
beam of electrons, but in this instrument the beam does not autoradiography. If a radioactive precursor of DNA (such
pass through the specimen (Figure 1–8b). Instead, the surface as tritium-labeled thymidine) is used, it is possible to know
of the specimen is first dried and spray-coated with a very thin which cells in a tissue (and how many) are replicating DNA
FIGURE 1–9â•‡ Microscopic autoradiography.
G
G
a b
Autoradiographs are tissue preparations in which particles called (a) Black grains of silver from the light-sensitive material coating
silver grains indicate the cells or regions of cells in which specific the specimen are visible over cell regions with secretory granules
macromolecules were synthesized just prior to fixation. Shown and the duct indicating glycoprotein locations. (X1500)
here are autoradiographs from the salivary gland of a mouse (b) The same tissue prepared for TEM autoradiography shows sil-
injected with 3H-fucose 8 hours before tissue fixation. Fucose was ver grains with a coiled or amorphous appearance again localized
incorporated into oligosaccharides, and the free 3H-fucose was mainly over the granules (G) and in the gland lumen (L). (X7500)
removed during fixation and sectioning of the gland. Autoradio- (Figure 1–9b, used with permission from Drs Ticiano G. Lima and
graphic processing and microscopy reveal locations of newly syn- A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.)
thesized glycoproteins containing that sugar.
and preparing to divide. Dynamic events may also be analyzed.

For example, if one wishes to know where in the cell protein is
›â•ºENZYME HISTOCHEMISTRY
produced, if it is secreted, and its path in the cell before being Enzyme histochemistry (or cytochemistry) is a method for
secreted, several animals are injected with a radioactive amino localizing cellular structures using a specific enzymatic activ-
acid and tissues collected at different times after the injections. ity present in those structures. To preserve the endogenous
Autoradiography of the tissues from the sequential times will enzymes histochemical procedures usually use unfixed or
indicate the migration of the radioactive proteins. mildly fixed tissue, which is sectioned on a cryostat to avoid
adverse effects of heat and organic solvents on enzymatic
›â•ºCELL & TISSUE CULTURE activity. For enzyme histochemistry (1) tissue sections are
immersed in a solution containing the substrate of the enzyme
Live cells and tissues can be maintained and studied outside to be localized; (2) the enzyme is allowed to act on its sub-
the body in culture (in vitro). In the organism (in vivo), cells strate; (3) the section is then put in contact with a marker
are bathed in fluid derived from blood plasma and containing compound that reacts with a product of the enzymatic action
many different molecules required for survival and growth. on the substrate; and (4) the final product from the marker,
Cell culture allows the direct observation of cellular behavior which must be insoluble and visible by light or electron
under a phase-contrast microscope and many experiments microscopy, precipitates over the site of the enzymes, identify-
technically impossible to perform in the intact animal can be ing their location.
accomplished in vitro. Examples of enzymes that can be detected histochemi-
The cells and tissues are grown in complex solutions of cally include the following:
known composition (salts, amino acids, vitamins) to which ■âŒ€ Phosphatases, which remove phosphate groups from
serum or specific growth factors are added. Cells to be cultured macromolecules (Figure 1–10).
are dispersed mechanically or enzymatically from a tissue or ■âŒ€ Dehydrogenases, which transfer hydrogen ions from
organ and placed with sterile procedures in a clear dish to one substrate to another, such as many enzymes of the
which they adhere, usually as a single layer (Figure 1–5). Such citric acid (Krebs) cycle, allowing histochemical identifi-
preparations are called primary cell cultures. Some cells can cation of such enzymes in mitochondria.
be maintained in vitro for long periods because they become ■âŒ€ Peroxidase, which promotes the oxidation of sub-
immortalized and constitute a permanent cell line. Most cells strates with the transfer of hydrogen ions to hydrogen
obtained from normal tissues have a finite, genetically pro- peroxide.
grammed life span. However certain changes (some related
to oncogenes; see Chapter 3) can promote cell immortality, a
process called transformation, and are similar to the initial › â•ºâ•º MEDICAL APPLICATION
changes in a normal cell’s becoming a cancer cell. Improve-
Many enzyme histochemical procedures are used in the
ments in culture technology and use of specific growth factors
medical laboratory, including Perls’ Prussian blue reaction for
now allow most cell types to be maintained in vitro.
iron (used to diagnose the iron storage diseases, hemochro-
As shown in Chapter 2, incubation of living cells in vitro
matosis and hemosiderosis), the PAS-amylase and alcian blue
with a variety of new fluorescent compounds that are seques-
reactions for polysaccharides (to detect glycogenosis and
tered and metabolized in specific compartments of the cell
mucopolysaccharidosis), and reactions for lipids and sphin-
provides a new approach to understanding these compart-
golipids (to detect sphingolipidosis).
ments both structurally and physiologically. Other histologic
techniques applied to cultured cells have been particularly
important for understanding the locations and functions of
microtubules, microfilaments, and other components of the
cytoskeleton. ›â•ºVISUALIZING SPECIFIC MOLECULES
A specific macromolecule present in a tissue section may also
be identified by using tagged compounds or macromolecules
› â•ºâ•º MEDICAL APPLICATION that bind specifically with the molecule of interest. The com-
Cell culture is very widely used to study molecular changes pounds that interact with the molecule must be visible with
that occur in cancer; to analyze infectious viruses, myco- the light or electron microscope, often by being tagged with a
plasma, and some protozoa; and for many routine genetic or detectible label. The most commonly used labels are fluores-
chromosomal analyses. Cervical cancer cells from a patient cent compounds, radioactive atoms that can be detected with
later identified as Henrietta Lacks, who died from the disease autoradiography, molecules of peroxidase or other enzymes
in 1951, were used to establish one of the first cell lines, that can be detected with histochemistry, and metal (usually
called HeLa cells, which are still used in research on cellular gold) particles that can be seen with light and electron micros-
structure and function throughout the world. copy. These methods can be used to detect and localize specific
sugars, proteins, and nucleic acids.
Visualizing Specific Molecules 11
Examples of molecules that interact specifically with

FIGURE 1–10â•‡ Enzyme histochemistry.
other molecules include the following:
C H A P T E R
■âŒ€ Phalloidin, a compound extracted from mushroom,
Amanita phalloides, interacts strongly with the actin pro-
tein of microfilaments.
L ■âŒ€ Protein A, purified from Staphylococcus aureus bacte-
ria, binds to the Fc region of antibody molecules, and
can therefore be used to localize naturally occurring or
1
applied antibodies bound to cell structures.
Histology & Its Methods of Study â•‡ ■â•‡ Visualizing Specific Molecules
■âŒ€ Lectins, glycoproteins derived mainly from plant seeds,
bind to carbohydrates with high affinity and specificity.
Different lectins bind to specific sugars or sequences of
sugar residues, allowing fluorescently labeled lectins to
be used to stain specific glycoproteins or other macro-
molecules bearing specific sequences of sugar residues.
Immunohistochemistry
A highly specific interaction between macromolecules is that
between an antigen and its antibody. For this reason labeled
antibodies are routinely used in immunohistochemistry
L L
to identify and localize many specific proteins, not just
those with enzymatic activity that can be demonstrated by
histochemistry.
aa The body’s immune cells interact with and produce anti-
bodies against other macromolecules—called antigens—that
are recognized as “foreign,” not a normal part of the organism,
and potentially dangerous. Antibodies belong to the immu-
noglobulin family of glycoproteins and are secreted by lym-
phocytes. These molecules normally bind specifically to their
Ly provoking antigens and help eliminate them.
Widely applied for both research and diagnostic pur-
poses, every immunohistochemical technique requires an
antibody against the protein that is to be detected. This means
Ly that the protein must have been previously purified using bio-
chemical or molecular methods so that antibodies against it
can be produced. To produce antibodies against protein x of a
certain animal species (eg, a human or rat), the isolated pro-
tein is injected into an animal of another species (eg, a rabbit
or a goat). If the protein’s amino acid sequence is sufficiently
b N different for this animal to recognize it as foreign—that is, as
an antigen—the animal will produce antibodies against the
protein.
(a) Micrograph of cross sections of kidney tubules treated Different groups (clones) of lymphocytes in the injected
histochemically to demonstrate alkaline phosphatases (with animal recognize different parts of protein x and each clone
maximum activity at an alkaline pH) showing strong activity of produces an antibody against that part. These antibodies are
this enzyme at the apical surfaces of the cells at the lumens (L)
of the tubules. (X200)
collected from the animal’s plasma and constitute a mixture
of polyclonal antibodies, each capable of binding a different
(b) TEM image of a kidney cell in which acid phosphatase has
been localized histochemically in three lysosomes (Ly) near the region of protein x.
nucleus (N). The dark material within these structures is lead It is also possible, however, to inject protein x into a
phosphate that precipitated in places with acid phosphatase mouse and a few days later isolate the activated lymphocytes
activity. (X25,000) and place them into culture. Growth and activity of these cells
(Figure 1–10b, used with permission from Dr Eduardo
can be prolonged indefinitely by fusing them with lymphocytic
Katchburian, Department of Morphology, Federal University of
São Paulo, Brazil.) tumor cells to produce hybridoma cells. Different hybridoma
clones produce different antibodies against the several parts
of protein x and each clone can be isolated and cultured sepa- The indirect method is used more widely in research and
rately so that the different antibodies against protein x can be pathologic tests because it is more sensitive, with the extra
collected separately. Each of these antibodies is a monoclo- level of antibody binding serving to amplify the visible signal.
nal antibody. An advantage to using a monoclonal antibody Moreover, the same preparation of labeled secondary antibody
rather than polyclonal antibodies is that it can be selected to can be used in studies with different primary antibodies (spe-
be highly specific and to bind strongly to the protein to be cific for different antigens) as long as all these are made in the
detected, with less nonspecific binding to other proteins that same species. There are other indirect methods that involve the
are similar to the one of interest. use of other intermediate molecules, such as the biotin-avidin
In immunohistochemistry a tissue section that one technique, which are also used to amplify detection signals.
believes contains the protein of interest is incubated in a solu- Examples of indirect immunocytochemistry are shown in
tion containing antibody (either monoclonal or polyclonal) Figure 1–12, demonstrating the use of this method with cells
against this protein. The antibody binds specifically to the in culture or after tissue sectioning for both light microscopy
protein and after a rinse the protein’s location in the tissue or and TEM.
cells can be seen with either the light or electron microscope
by visualizing the antibody. Antibodies are commonly tagged › â•ºâ•º MEDICAL APPLICATION
with fluorescent compounds, with peroxidase or alkaline
Because cells in some diseases, including many cancer cells,
phosphatase for histochemical detection, or with electron-
often produce proteins unique to their pathologic condition,
dense gold particles for TEM.
immunohistochemistry can be used by pathologists to diag-
As Figure 1–11 indicates, there are direct and indirect
nose many diseases, including certain types of tumors and
methods of immunocytochemistry. The direct method just
some virus-infected cells. Table 1-1 shows some applications
involves a labeled antibody that binds the protein of interest.
of immunocytochemistry routinely used in clinical practice.
Indirect immunohistochemistry involves sequential
application of two antibodies and additional washing steps. The
(primary) antibody specifically binding the protein of interest Hybridization Techniques
is not labeled. The detectible tag is conjugated to a second- Hybridization usually implies the specific binding between
ary antibody made in an animal species different (“foreign”) two single strands of nucleic acid, which occurs under appro-
from that which made the primary antibody. For example, pri- priate conditions if the strands are complementary. The greater
mary antibodies made by mouse lymphocytes (such as most the similarities of their nucleotide sequences, the more read-
monoclonal antibodies) are specifically recognized and bound ily the complementary strands form “hybrid” double-strand
by antibodies made in a rabbit or goat injected with mouse molecules. Hybridization at stringent conditions allows the
antibody immunoglobulin. specific identification of sequences in genes or RNA. This can
FIGURE 1–11â•‡ Immunocytochemistry techniques.
Labeled
secondary
Labeled Unlabeled antibody
antibody primary
antibody
Antigen Antigen
Tissue section
Glass slide
Direct Indirect
Immunocytochemistry (or immunohistochemistry) can be direct labeled secondary antibody is obtained that was (1) made in
or indirect. Direct immunocytochemistry (left) uses an antibody another species against immunoglobulin proteins (antibodies)
made against the tissue protein of interest and tagged directly from the species in which the primary antibodies were made and
with a label such as a fluorescent compound or peroxidase. When (2) labeled with a fluorescent compound or peroxidase. When
placed with the tissue section on a slide, these labeled antibod- the labeled secondary antibody is applied to the tissue section, it
ies bind specifically to the protein (antigen) against which they specifically binds the primary antibodies, indirectly labeling the
were produced and can be visualized by the appropriate method. protein of interest on the slide. Because more than one labeled
Indirect immunocytochemistry (right) uses first a primary secondary antibody can bind each primary antibody molecule,
antibody made against the protein (antigen) of interest and labeling of the protein of interest is amplified by the indirect
applied to the tissue section to bind its specific antigen. Then a method.
Visualizing Specific Molecules 13
FIGURE 1–12â•‡ Cells and tissues stained by immunohistochemistry.
1 C H A P T E R
Histology & Its Methods of Study â•‡ ■â•‡ Visualizing Specific Molecules
a c
throughout the cytoplasm. Primary antibodies against the

filament protein desmin and fluorescein isothiocyanate
(FITC)–labeled secondary antibodies were used in the indirect
staining technique, with the nucleus counterstained blue with
DAPI. (X650)
(b) A section of small intestine treated with an antibody against
the enzyme lysozyme. The secondary antibody labeled with
peroxidase was then applied and the localized brown color
produced histochemically with the peroxidase substrate
3,3′-diamino-azobenzidine (DAB). The method demonstrates
lysozyme-containing structures in scattered macrophages and in
the large clusters of cells. Nuclei were counterstained with hema-
toxylin. (X100)
(c) A section of pancreatic cells in a TEM preparation incubated
with an antibody against the enzyme amylase and then with
protein A coupled with gold particles. Protein A has high affin-
ity toward antibody molecules and the resulting image reveals
b
the presence of amylase with the gold particles localized as very
small black dots over dense secretory granules and developing
granules (left). With specificity for immunoglobulin molecules,
Immunocytochemical methods to localize specific proteins can labeled protein A can be used to localize any primary antibody.
be applied to either light microscopic or TEM preparations using a (X5000)
variety of labels. (Figure 1–12c, used with permission from Dr Moise Bendayan,
(a) A single cultured uterine cell stained fluorescently Departments of Pathology and Cell Biology, University of Montreal,
to reveal a meshwork of intermediate filaments (green) Montreal, Canada.)
â•‡
TABLE 1-1 Examples of specific antigens with diagnostic importance.
Antigens Diagnosis
Specific cytokeratins Tumors of epithelial origin

Protein and polypeptide hormones Certain endocrine tumors
Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast
Steroid hormone receptors Breast duct cell tumors
Antigens produced by viruses Specific virus infections
occur with cellular DNA or RNA when nucleic acid sequences › â•ºâ•º MEDICAL APPLICATION
in solution are applied directly to prepared cells and tissue sec-
Warts on the skin of the genitals and elsewhere are due to
tions, a procedure called in situ hybridization (ISH).
infection with the human papilloma virus (HPV) which causes
This technique is ideal for (1) determining if a cell has
the characteristic benign proliferative growth. As shown in
a specific sequence of DNA, such as a gene or part of a gene
Figure 1–12 such virus-infected cells can often be demon-
(Figure 1–13), (2) identifying the cells containing specific
strated by ISH. Certain cancer cells with unique or elevated
messenger RNAs (mRNAs) (in which the corresponding
expression of specific genes are also localized in tumors and
gene is being transcribed), or (3) determining the localiza-
studied microscopically by ISH.
tion of a gene in a specific chromosome. DNA and RNA of
the cells must be initially denatured by heat or other agents
to become completely single-stranded and the nucleotide
sequences of interest are detected with probes consisting of
single-stranded complementary DNA (cDNA). The probe
›â•ºINTERPRETATION OF STRUCTURES
IN TISSUE SECTIONS
may be obtained by cloning, by polymerase chain reaction
(PCR) amplification of the target sequence, or by chemical In studying and interpreting stained tissue sections, it is
synthesis if the desired sequence is short. The probe is tagged important to remember that microscopic preparations are the
with nucleotides containing a radioactive isotope (localized by end result of a series of processes that began with collecting
autoradiography) or modified with a small compound such as the tissue and ended with mounting a coverslip on the slide.
digoxigenin (identified by immunocytochemistry). A solution Certain steps in this procedure may distort the tissues slightly,
containing the probe is placed over the specimen under con- producing minor structural abnormalities called artifacts not
ditions allowing hybridization and after the excess unbound present in the living tissue.
probe is washed off, the localization of the hybridized probe is One such distortion is minor shrinkage of cells or tissue
revealed through its label. regions produced by the fixative, by the ethanol, or by the heat
needed for paraffin embedding. Shrinkage can create artificial
spaces between cells and other tissue components. Such spaces
FIGURE 1–13â•‡ In situ hybridization (ISH). can also result from the loss of lipids or low-molecular-weight
substances not preserved by the fixative or removed by the
dehydrating and clearing fluids. Slight cracks in sections may
also appear as large spaces in the tissue.
Other artifacts may include small wrinkles in the section
(which the novice may confuse with linear structures in tissue)
and precipitates from the stain (which may be confused with
cellular structures such as cytoplasmic granules). Students must
be aware of the existence of artifacts and able to recognize them.
Another difficulty in the study of histologic sections is the
impossibility of differentially staining all tissue components on
one slide. A single stain can seldom demonstrate well nuclei,
mitochondria, lysosomes, basement membranes, elastic fibers,
etc. With the light microscope, it is necessary to examine prepa-
rations stained by different methods before an idea of the whole
composition and structure of a cell or tissue can be obtained. The
TEM allows the observation of cells with all its internal struc-
tures and surrounding ECM components, but only a few cells in
a tissue can be conveniently studied in these very small samples.
Finally, when a structure’s three-dimensional volume is
cut into very thin sections, the sections appear microscopically
to have only two dimensions: length and width. When examin-
In situ hybridization of this tissue section with probes for the ing a section under the microscope, the viewer must always keep
human papilloma virus (HPV) reveals the presence of many
cells containing the virus. The section was incubated with a
in mind that components are missing in front of and behind
solution containing a digoxigenin-labeled complementary what is being seen because many tissue structures are thicker
DNA (cDNA) probe for the HPV DNA. The probe was then than the section. Round structures seen microscopically may
visualized by direct immunohistochemistry using peroxidase- actually be portions of spheres or tubes. Because structures in
labeled antibodies against digoxigenin. This procedure stains a tissue have different orientations, their two-dimensional (2D)
brown only those cells containing HPV. (X400; H&E)
(Used with permission from Dr Jose E. Levi, Virology Lab,
appearance will also vary depending on the plane of section. A
Institute of Tropical Medicine, University of São Paulo, Brazil.) single convoluted tube will appear in a tissue section as many
separate rounded or oval structures (Figure 1–14).
Interpretation of Structures in Tissue Sections 15
FIGURE 1–14â•‡ Interpretation of 3D structures in 2D sections.
1 C H A P T E R
Histology & Its Methods of Study â•‡ ■â•‡ Interpretation of Structures in Tissue Sections
In thin sections 3D structures appear to have only two dimensions.
Such images must be interpreted correctly to understand the actual
structure of tissue and organ components. For example, blood ves-
sels and other tubular structures appear in sections as round or oval
shapes whose size and shape depend on the transverse or oblique
angle of the cut. A highly coiled tube will appear as several round
and oval structures. In TEM sections of cells, round structures may
represent spherical organelles or transverse cuts through tubular
organelles such as mitochondria. It is important to develop such
interpretive skill to understand tissue and cell morphology in micro-
scopic preparations.
Histology & Its Methods of Studyâ•‡ SUMMARY OF KEY POINTS
Preparation of Tissues for Study Autoradiography

■âŒ€ Chemical fixatives such as formalin are used to preserve tissue ■âŒ€ This process localizes cell components synthesized using radioactive
structure by cross-linking and denaturing proteins, inactivating precursors by detecting silver grains produced by weakly emitted
enzymes, and preventing cell autolysis or self-digestion. radiation in a photographic emulsion coating the tissue section or cells.
■âŒ€ Dehydration of the fixed tissue in alcohol and clearing in organic ■âŒ€ With either light microscopy or TEM, autoradiography permits
solvents prepare it for embedding and sectioning. unique studies of processes such as tissue growth (using radioactive
■âŒ€ Embedding in paraffin wax or epoxy resin allows the tissue to be DNA precursors) or cellular pathways of macromolecular synthesis.
cut into very thin sections (slices) with a microtome.
■âŒ€ Sections are mounted on glass slides for staining, which is required to Cell & Tissue Culture
reveal specific cellular and tissue components with the microscope. ■âŒ€ Cells can be grown in vitro from newly explanted tissues (primary
■âŒ€ The most commonly used staining method is a combination of the cultures) or as long-established cell lines and can be examined in the
stains hematoxylin and eosin (H&E), which act as basic and acidic living state by phase-contrast light microscopy.
dyes, respectively. Enzyme Histochemistry
■âŒ€ Cell substances with a net negative (anionic) charge, such as DNA
and RNA, react strongly with hematoxylin and basic stains; such ■âŒ€ Histochemical (or cytochemical) techniques use specific enzy-
material is said to be “basophilic.” matic activities in lightly fixed or unfixed tissue sections to produce
■âŒ€ Cationic substances, such as collagen and many cytoplasmic proteins visible products in the specific enzyme locations.
react with eosin and other acidic stains and are said to be “acidophilic.” ■âŒ€ Fixation and paraffin embedding denatures most enzymes, so histo-
chemistry usually uses frozen tissue sectioned with a cryostat.
Light Microscopy ■âŒ€ Enzyme classes for which histochemical study is useful include
■âŒ€ Bright-field microscopy, the method most commonly used by phosphatases, dehydrogenases, and peroxidases, with peroxidase
both students and pathologists, uses ordinary light and the colors often conjugated to antibodies used in immunohistochemistry.
are imparted by tissue staining. Visualizing Specific Molecules
■âŒ€ Fluorescence microscopy uses ultraviolet light, under which only
fluorescent molecules are visible, allowing localization of fluores- ■âŒ€ Some substances specifically bind certain targets in cells.
cent probes which can be much more specific than routine stains. ■âŒ€ Immunohistochemistry is based on specific reactions between an
■âŒ€ Phase-contrast microscopy uses the differences in refractive antigen and antibodies labeled with visible markers, often fluores-
index of various natural cell and tissue components to produce an cent compounds or peroxidase for light microscopy and gold par-
image without staining, allowing observation of living cells. ticles for TEM.
■âŒ€ Confocal microscopy involves scanning the specimen at succes- ■âŒ€ If the cell or tissue antigen of interest is detected by directly binding
sive focal planes with a focused light beam, often from a laser, and a labeled primary antibody specific for that antigen, the process is
produces a 3D reconstruction from the images. considered direct immunohistochemistry.
■âŒ€ Indirect immunohistochemistry uses an unlabeled primary anti- Interpretation of Structures in Tissue Sections
body that is detected bound to its antigen with labeled secondary ■âŒ€ Many steps in tissue processing, slide preparation, and staining can
antibodies. introduce minor artifacts such as spaces and precipitates that are
■âŒ€ The indirect immunohistochemical method is more commonly not normally present in the living tissue and must be recognized.
used because the added level of antibody binding amplifies the sig- ■âŒ€ Sections of cells or tissues are essentially 2D planes through 3D
nal detected and provides greater technical flexibility. structures, and understanding this fact is important for their cor-
■âŒ€ Specific gene sequences or mRNAs of cells can be detected micro- rect interpretation and study.
scopically using labeled complementary DNA (cDNA) probes in a
technique called in situ hybridization (ISH).
Histology & Its Methods of Studyâ•‡ ASSESS YOUR KNOWLEDGE
1. In preparing tissue for routine light microscopic study, which 7. Microscopic autoradiography uses radioactivity and can be employed
procedure immediately precedes clearing the specimen with an to study what features in a tissue section?
organic solvent? a. The types of enzymes found in various cell locations
a. Dehydration b. Cellular sites where various macromolecules are synthesized
b. Fixation c. The sequences of mRNA made in the cells
c. Staining d. The dimensions of structures within the cells
d. Clearing e. The locations of genes transcribed for specific mRNA
e. Embedding
8. To identify and localize a specific protein within cells or the extracel-
2. Which of the following staining procedures relies on the cationic lular matrix one would best use what approach?
and anionic properties of the material to be stained? a. Autoradiography
a. Enzyme histochemistry b. Enzyme histochemistry
b. Periodic acid-Schiff reaction c. Immunohistochemistry
c. Hematoxylin & eosin staining d. Transmission electron microscopy
d. Immunohistochemistry e. Polarizing microscopy
e. Metal impregnation techniques
9. In situ hybridization is a histological technique used to visualize what
3. In a light microscope used for histology, resolution and magnifica- type of macromolecule?
tion of cells are largely dependent on which component? a. Proteins
a. Condenser b. Carbohydrates
b. Objective lens c. Certain enzymes
c. Eyepieces or ocular lenses d. Nucleic acids
d. Specimen slide e. Lipids
e. The control for illumination intensity
10. Hospital laboratories frequently use unfixed, frozen tissue specimens
4. Cellular storage deposits of glycogen, a free polysaccharide, could sectioned with a cryostat for rapid staining, microscopic examina-
best be detected histologically using what procedure? tion, and diagnosis of pathological conditions. Besides saving much
a. Autoradiography time by avoiding fixation and procedures required for paraffin
b. Electron microscopy embedding, frozen sections retain and allow study of what macro-
c. Enzyme histochemistry molecules normally lost in the paraffin procedure?
d. Hematoxylin & eosin staining a. Carbohydrates
e. Periodic acid-Schiff reaction b. Small mRNA
c. Basic proteins
5. Adding heavy metal compounds to the fixative and ultrathin sec- d. Acidic proteins
tioning of the embedded tissue with a glass knife are techniques e. Lipids
used for which histological procedure?
a. Scanning electron microscopy
b. Fluorescent microscopy
c. Enzyme histochemistry
d. Confocal microscopy
e. Transmission electron microscopy
6. Resolution in electron microscopy greatly exceeds that of light
microscopy due to which of the following?
a. The wavelength of the electrons in the microscope beam is
shorter than that of a beam of light.
b. The lenses of an electron microscope are of greatly improved
quality.
c. For electron microscopy the tissue specimen does not require
staining.
d. The electron microscope allows much greater magnification of
a projected image than a light microscope provides.
e. An electron microscope can be much more finely controlled
than a light microscope.
Answers: 1a, 2c, 3b, 4e, 5e, 6a, 7b, 8c, 9d, 10e
C H A P T E R
CELL DIFFERENTIATION
2 The Cytoplasm
17 Proteasomes
Mitochondria
37
38
THE PLASMA MEMBRANE 17
Peroxisomes 39
Transmembrane Proteins & Membrane Transport 19
Vesicular Transport: Endocytosis & Exocytosis 21 THE CYTOSKELETON 42
Signal Reception & Transduction 26 Microtubules 43
CYTOPLASMIC ORGANELLES 27 Microfilaments (Actin Filaments) 44
Intermediate Filaments 45
Ribosomes 27
Endoplasmic Reticulum 28 INCLUSIONS 47
Golgi Apparatus 31 SUMMARY OF KEY POINTS 51
Secretory Granules 33
ASSESS YOUR KNOWLEDGE 52
Lysosomes 34
C ells and extracellular material together comprise all

the tissues that make up the organs of multicellular
animals. In all tissues, cells themselves are the basic
structural and functional units, the smallest living parts of the
body. Animal cells are eukaryotic, with distinct membrane-
Major cellular functions performed by specialized cells
in the body are listed in Table 2–1. It is important to under-
stand that the functions listed there can be performed by most
cells of the body; specialized cells have greatly expanded their
capacity for one or more of these functions during differen-
limited nuclei surrounded by cytoplasm which contains vari- tiation. Changes in cells’ microenvironments under normal
ous membrane-limited organelles and the cytoskeleton. In and pathologic conditions can cause the same cell type to have
contrast, the smaller prokaryotic cells of bacteria typically variable features and activities. Cells that appear similar struc-
have a cell wall around the plasmalemma and lack nuclei and turally often have different families of receptors for signaling
membranous cytoplasmic structures. molecules such as hormones and extracellular matrix (ECM)
components, causing them to behave differently. For example,
because of their diverse arrays of receptors, breast fibroblasts
›â•ºCELL DIFFERENTIATION and uterine smooth muscle cells are exceptionally sensitive to
female sex hormones while most other fibroblasts and smooth
The human organism consists of hundreds of different cell
muscle cells are insensitive.
types, all derived from the zygote, the single cell formed
by the merger of a spermatozoon with an oocyte at fertil-
ization. The first zygotic cellular divisions produce cells
called blastomeres, and as part of the early embryo’s inner ›â•ºTHE PLASMA MEMBRANE
cell mass blastomeres give rise to all tissue types of the fetus. The plasma membrane (cell membrane or plasmalemma)
Explanted to tissue culture cells of the inner cell mass are that envelops every eukaryotic cell consists of phospholipids,
called embryonic stem cells. Most cells of the fetus undergo cholesterol, and proteins, with oligosaccharide chains cova-
a specialization process called differentiation in which lently linked to many of the phospholipid and protein mol-
they differentially express sets of genes that mediate specific ecules. This limiting membrane functions as a selective barrier
cytoplasmic activities, becoming very efficient in specialized regulating the passage of materials into and out of the cell and
functions and usually changing their shape accordingly. For facilitating the transport of specific molecules. One important
example, muscle cell precursors elongate into fiber-like cells role of the cell membrane is to keep constant the ion content
containing large arrays of actin and myosin. All animal cells of cytoplasm, which differs from that of the extracellular fluid.
contain actin filaments and myosins, but muscle cells are spe- Membranes also carry out a number of specific recognition
cialized for using these proteins to convert chemical energy and signaling functions, playing a key role in the interactions
into forceful contractions. of the cell with its environment.
17
18 CHAPTER 2â•… ■â•… The Cytoplasm
abundant in the outer half, while phosphatidylserine and

Differentiated cells typically
TABLE 2–1 specialize in one activity.
phosphatidylethanolamine are more concentrated in the inner
â•‡
layer. Some of the outer layer’s lipids, known as glycolipids,
General Cellular Activity Specialized Cell(s) include oligosaccharide chains that extend outward from the
cell surface and contribute to a delicate cell surface coating
Movement Muscle and other contractile called the glycocalyx (Figures 2–1b and 2–2). With the trans-
cells mission electron microscope (TEM) the cell membrane—as
Form adhesive and tight Epithelial cells well as all cytoplasmic membranes—may exhibit a trilaminar
junctions between cells appearance after fixation in osmium tetroxide; osmium binds
Synthesize and secrete Fibroblasts, cells of bone and the polar heads of the phospholipids and the oligosaccharide
components of the extracellular cartilage chains, producing the two dark outer lines that enclose the
matrix light band of osmium-free fatty acids (Figure 2–1b).
Convert physical and chemical Neurons and sensory cells Proteins are major constituents of membranes (~50%
stimuli into action potentials by weight in the plasma membrane). Integral proteins
Synthesis and secretion of Cells of digestive glands are incorporated directly within the lipid bilayer, whereas
degradative enzymes peripheral proteins are bound to one of the two membrane
Synthesis and secretion of Cells of mucous glands
surfaces, particularly on the cytoplasmic side (Figure 2–2).
glycoproteins Peripheral proteins can be extracted from cell membranes
with salt solutions, whereas integral proteins can be extracted
Synthesis and secretion of Certain cells of the adrenal
steroids gland, testis, and ovary
only by using detergents to disrupt the lipids. The polypeptide
chains of many integral proteins span the membrane, from
Ion transport Cells of the kidney and salivary one side to the other, several times and are accordingly called
gland ducts
multipass proteins. Integration of the proteins within the
Intracellular digestion Macrophages and neutrophils lipid bilayer is mainly the result of hydrophobic interactions
Lipid storage Fat cells between the lipids and nonpolar amino acids of the proteins.
Freeze-fracture electron microscope studies of mem-
Metabolite absorption Cells lining the intestine
branes show that parts of many integral proteins protrude
from both the outer or inner membrane surface (Figure 2–2b).
Although the plasma membrane defines the outer limit of Like those of glycolipids, the carbohydrate moieties of glyco-
the cell, a continuum exists between the interior of the cell and proteins project from the external surface of the plasma mem-
extracellular macromolecules. Certain plasma membrane pro- brane and contribute to the glycocalyx (Figure 2–3). They are
teins, the integrins, are linked to both the cytoskeleton and important components of proteins acting as receptors, which
ECM components and allow continuous exchange of influ- participate in important interactions such as cell adhesion, cell
ences, in both directions, between the cytoplasm and material recognition, and the response to protein hormones. As with
in the ECM. lipids, the distribution of membrane polypeptides is different
Membranes range from 7.5 to 10 nm in thickness and in the two surfaces of the cell membranes. Therefore, all mem-
consequently are visible only in the electron microscope. The branes in the cell are asymmetric.
line between adjacent cells sometimes seen faintly with the Studies with labeled membrane proteins of cultured cells
light microscope consists of plasma membrane proteins plus reveal that many such proteins are not bound rigidly in place
extracellular material, which together can reach a dimension and are able to move laterally (Figure 2–4). Such observations as
visible by light microscopy. well as data from biochemical, electron microscopic, and other
Membrane phospholipids are amphipathic, consisting of studies showed that membrane proteins comprise a moveable
two nonpolar (hydrophobic or water-repelling) long-chain mosaic within the fluid lipid bilayer, the well-established fluid
fatty acids linked to a charged polar (hydrophilic or water- mosaic model for membrane structure (Figure 2–2a). Unlike
attracting) head that bears a phosphate group (Figure 2–1a). the lipids, however, lateral diffusion of many membrane pro-
Phospholipids are most stable when organized into a double teins is often restricted by their cytoskeletal attachments.
layer (bilayer) with the hydrophobic fatty acid chains located Moreover, in most epithelial cells tight junctions between the
in a middle region away from water and the hydrophilic polar cells (see Chapter 4) also restrict lateral diffusion of unattached
head groups contacting the water (Figure 2–1b). Molecules transmembrane proteins and outer layer lipids, producing dif-
of cholesterol, a sterol lipid, insert at varying densities among ferent domains within the cell membranes.
the closely-packed phospholipid fatty acids, restricting their Membrane proteins that are components of large enzyme
movements and modulating the fluidity of all membrane complexes are also usually less mobile, especially those
components. The phospholipids in each half of the bilayer are involved in the transduction of signals from outside the cell.
different. For example, in the well-studied membranes of red Such protein complexes are located in specialized membrane
blood cells phosphatidylcholine and sphingomyelin are more patches termed lipid rafts with higher concentrations of
The Plasma Membrane 19
FIGURE 2–1â•‡ Lipids in membrane structure.
C H A P T E R
Polar head group Nonpolar fatty acid chains
(hydrophilic) (hydrophobic)
O
Saturated CH3
CH2 O C
fatty acid
O
2
(straight) CH3
CH O C
The Cytoplasmâ•‡ ■â•‡ The Plasma Membrane

O
Unsaturated
CH2 O P O X fatty acid OH
O– (bent)
General structure of a phospholipid Cholesterol
Sugar chains of a glycolipid
Phospholipids
Hydrophilic surface
Hydrophobic region
Extracellular fluid
Hydrophilic surface
Cholesterol
Cytoplasm
(a) Membranes of animal cells have as their major lipid com- throughout the lipid bilayer; cholesterol affects the packing of the
ponents phospholipids and cholesterol. A phospholipid is fatty acid chains, with a major effect on membrane fluidity. The
amphipathic, with a phosphate group charge on the polar head outer layer of the cell membrane also contains glycolipids with
and two long, nonpolar fatty acid chains, which can be straight extended carbohydrate chains.
(saturated) or kinked (at an unsaturated bond). Membrane choles- Sectioned, osmium-fixed cell membrane may have a faint trilam-
terol is present in about the same amount as phospholipid. inar appearance with the transmission electron microscope (TEM),
(b) The amphipathic nature of phospholipids produces the bilayer showing two dark (electron-dense) lines enclosing a clear (electron-
structure of membranes as the charged (hydrophilic) polar heads lucent) band. Reduced osmium is deposited on the hydrophilic
spontaneously form each membrane surface, in direct contact phosphate groups present on each side of the internal region of
with water, and the hydrophobic nonpolar fatty acid chains are fatty acid chains where osmium is not deposited. The “fuzzy” mate-
buried in the membrane’s middle, away from water. Cholesterol rial on the outer surface of the membrane represents the glycocalyx
molecules are also amphipathic and are interspersed less evenly of oligosaccharides of glycolipids and glycoproteins. (X100,000)
cholesterol and saturated fatty acids which reduce lipid fluid- molecules cross the membrane by the general mechanisms
ity. This together with the presence of scaffold proteins that shown schematically in Figure 2–5 and explained as follows:
maintain spatial relationships between enzymes and signal-
ing proteins allows the proteins assembled within lipid rafts to
■âŒ€ Diffusion transports small, nonpolar molecules directly
through the lipid bilayer. Lipophilic (fat-soluble) mol-
remain in close proximity and interact more efficiently. ecules diffuse through membranes readily, water very
slowly.
Transmembrane Proteins & MembraneTransport ■âŒ€ Channels are multipass proteins forming transmembrane
The plasma membrane is the site where materials are pores through which ions or small molecules pass selec-
exchanged between the cell and its environment. Most small tively. Cells open and close specific channels for Na+,
FIGURE 2–2â•‡ Proteins associated with the membrane lipid bilayer.
Sugar chain Sugar chain

of glycolipid of glycoprotein
2 E face
Peripheral protein 1
Transmembrane protein
Lipid
P face
(a) The fluid mosaic model of membrane structure emphasizes center. Splitting occurs along the line of weakness formed by
that the phospholipid bilayer of a membrane also contains pro- the fatty acid tails of phospholipids. Electron microscopy of such
teins inserted in it or associated with its surface (peripheral pro- cryofracture preparation replicas provides a useful method for
teins) and that many of these proteins move within the fluid lipid studying membrane structures. Most of the protruding mem-
phase. Integral proteins are firmly embedded in the lipid layers; brane particles seen (1) are proteins or aggregates of proteins
those that completely span the bilayer are called transmembrane that remain attached to the half of the membrane adjacent to
proteins. Hydrophobic amino acids of these proteins interact with the cytoplasm (P or protoplasmic face). Fewer particles are found
the hydrophobic fatty acid portions of the membrane lipids. Both attached to the outer half of the membrane (E or extracellular
the proteins and lipids may have externally exposed oligosaccha- face). Each protein bulging on one surface has a corresponding
ride chains. depression (2) on the opposite surface.
(b) When cells are frozen and fractured (cryofracture), the lipid
bilayer of membranes is often cleaved along the hydrophobic
K+, Ca2+ and other ions in response to various physio- down a concentration gradient due to its kinetic energy. In
logical stimuli. Water molecules usually cross the plasma contrast, membrane pumps are enzymes engaged in active
membrane through channel proteins called aquaporins. transport, utilizing energy from the hydrolysis of adenos-
■âŒ€ Carriers are transmembrane proteins that bind small ine triphosphate (ATP) to move ions and other solutes across
molecules and translocate them across the membrane via membranes, against often steep concentration gradients.
conformational changes. Because they consume ATP pumps they are often referred to
as ATPases.
Diffusion, channels, and carrier proteins operate pas- These transport mechanisms are summarized with addi-
sively, allowing movement of substances across membranes tional detail in Table 2–2.
FIGURE 2–3â•‡ Membrane proteins.
C H A P T E R
Interstitial fluid
Phospholipid
2
Glycolipid Carbohydrate

Polar head of
phospholipid
molecule
Phospholipid
bilayer
Glycoprotein
Nonpolar tails
of phospholipid Cholesterol Protein
molecule
Integral protein
Peripheral protein
Filaments of
cytoskeleton
Cytosol
Functions of Plasma Membrane
1. Physical barrier: Establishes a flexible boundary, protects cellular contents, 3. Electrochemical gradients: Establishes and maintains an electrical
and supports cell structure. Phospholipid bilayer separates substances charge difference across the plasma membrane.
inside and outside the cell. 4. Communication: Contains receptors that recognize and respond to
2. Selective permeability: Regulates entry and exit of ions, nutrients, molecular signals.
and waste molecules through the membrane.
Both protein and lipid components often have covalently connections, and as selective gateways for molecules entering
attached oligosaccharide chains exposed at the external mem- the cell.
brane surface. These contribute to the cell’s glycocalyx, which Transmembrane proteins often have multiple hydrophobic
provides important antigenic and functional properties to the regions buried within the lipid bilayer to produce a channel or
cell surface. Membrane proteins serve as receptors for vari- other active site for specific transfer of substances through the
ous signals coming from outside cells, as parts of intercellular membrane.
Vesicular â•›Transport: â•›Endocytosis & â•›Exocytosis which project from the cell in a process dependent on
cytoskeletal changes. Fusion of the membranous folds
Macromolecules normally enter cells by being enclosed within
encloses the bacterium in an intracellular vacuole called
folds of plasma membrane (often after binding specific mem-
a phagosome, which then merges with a lysosome
brane receptors) which fuse and pinch off internally as cyto-
for degradation of its contents as discussed later in this
plasmic vesicles (or vacuoles) in a general process known as
chapter.
endocytosis. Three major types of endocytosis are recog-
nized, as summarized in Table 2–2 and Figure 2–6. 2. Pinocytosis (“cell drinking”) involves smaller invagina-
tions of the cell membrane which fuse and entrap extra-
1. Phagocytosis (“cell eating”) is the ingestion of particles cellular fluid and its dissolved contents. The resulting
such as bacteria or dead cell remnants. Certain blood- pinocytotic vesicles (~80 nm in diameter) then pinch
derived cells, such as macrophages and neutrophils, are off inwardly from the cell surface and either fuse with
specialized for this activity. When a bacterium becomes lysosomes or move to the opposite cell surface where
bound to the surface of a neutrophil, it becomes sur- they fuse with the membrane and release their contents
rounded by extensions of plasmalemma and cytoplasm outside the cell. The latter process, called transcytosis,
hormones, are integral membrane proteins at the cell

FIGURE 2–4â•‡ Experiment demonstrating the
surface. High-affinity binding of such ligands to their
fluidity of membrane proteins.
receptors causes these proteins to aggregate in special
membrane regions that then invaginate and pinch off
internally as vesicles.
The formation and fate of vesicles in receptor-mediated
endocytosis also often depend on specific peripheral proteins
on the cytoplasmic side of the membrane (Figure 2–7). The
occupied cell-surface receptors associate with these cyto-
plasmic proteins and begin invagination as coated pits.
The electron-dense coating on the cytoplasmic surface of
a such pits contains several polypeptides, the major one being
clathrin. Clathrin molecules interact like the struts of a
geodesic dome, forming that region of cell membrane into
a cage-like invagination that is pinched off in the cytoplasm
as a coated vesicle (Figure 2–7b) with the receptor-bound
ligands inside. Another type of receptor-mediated endocy-
tosis very prominent in endothelial cells produces invagina-
tions called caveolae (L. caveolae, little caves) that involve
the membrane protein caveolin.
In all these endocytotic processes, the vesicles or vacu-
oles produced quickly enter and fuse with the endosomal
b compartment, a dynamic collection in the peripheral cyto-
plasm of membranous tubules and vacuoles (Figure 2–7). The
clathrin molecules separate from the coated vesicles and recycle
back to the cell membrane for the formation of new coated
pits. Vesicle trafficking through the endosomal compartment
is directed largely through peripheral membrane G proteins
called Rab proteins, small GTPases that bind guanine nucle-
otides and associated proteins.
As shown in Figure 2–7, phagosomes and pinocytotic
vesicles typically fuse with lysosomes within the endosomal
compartment for digestion of their contents, while molecules
entering by receptor-mediated endocytosis may be directed
down other pathways. The membranes of many late endo-
somes have ATP-driven H+ pumps that acidify their interior,
activating the hydrolytic enzymes of lysosomes and in other
c endosomes causing ligands to uncouple from their receptors,
after which the two molecules are sorted into separate endo-
(a) Two types of cells were grown in tissue cultures, one with somes. The receptors may be sorted into recycling endosomes
fluorescently labeled transmembrane proteins in the plasma- and returned to the cell surface for reuse. Low-density lipopro-
lemma (right) and one without.
tein receptors, for example, are recycled several times within
(b) Cells of each type were fused together experimentally into
cells. Other endosomes may release their entire contents at a
hybrid cells.
separate domain of the cell membrane (transcytosis), which
(c) Minutes after the fusion of the cell membranes, the fluo-
rescent proteins of the labeled cell spread to the entire surface
occurs in many epithelial cells.
of the hybrid cells. Such experiments provide important data Movement of large molecules from inside to outside
supporting the fluid mosaic model. However, many mem- the cell usually involves vesicular transport in the process
brane proteins show more restricted lateral movements, being of exocytosis. In exocytosis a cytoplasmic vesicle containing
anchored in place by links to the cytoskeleton. the molecules to be secreted fuses with the plasma membrane,
resulting in the release of its contents into the extracellular space
without compromising the integrity of the plasma membrane
accomplishes bulk transfer of dissolved substances across (see “Transcytosis” in Figure 2–7a). Exocytosis is triggered in
the cell. many cells by transient increase in cytosolic Ca2+. Membrane
3. Receptor-mediated endocytosis: Receptors for many fusion during exocytosis is highly regulated, with selective
substances, such as low-density lipoproteins and protein interactions between several specific membrane proteins.
FIGURE 2–5â•‡ Major mechanisms by which molecules cross membranes.
2 C H A P T E R
(a) Simple diffusion (b) Channel (c) Carrier/pump
Lipophilic and some small, uncharged molecules can cross mem- conformations and release the molecule to the other side of the
branes by simple diffusion (a). membrane.
Most ions cross membranes in multipass proteins called channels Diffusion, channels and most carrier proteins translocate sub-
(b) whose structures include transmembrane ion-specific pores. stances across membranes using only kinetic energy. In contrast,
Many other larger, water-soluble molecules require binding pumps are carrier proteins for active transport of ions or other
to sites on selective carrier proteins (c), which then change their solutes and require energy derived from ATP.
Exocytosis of macromolecules made by cells occurs via are called exosomes, which can fuse with other cells transfer-
either of two pathways: ring their contents and membranes.
■âŒ€ Constitutive secretion is used for products that are
released from cells continuously, as soon as synthesis is Signal Reception & Transduction
complete, such as collagen subunits for the ECM. Cells in a multicellular organism communicate with one
■âŒ€ Regulated secretion occurs in response to signals com- another to regulate tissue and organ development, to control
ing to the cells, such as the release of digestive enzymes their growth and division, and to coordinate their functions.
from pancreatic cells in response to specific stimuli. Many adjacent cells form communicating gap junctions that
Regulated exocytosis of stored products from epithelial couple the cells and allow exchange of ions and small mol-
cells usually occurs specifically at the apical domains of ecules (see Chapter 4).
cells, constituting a major mechanism of glandular secretion Cells also use about 25 families of receptors to detect
(see Chapter 4). and respond to various extracellular molecules and physical
stimuli. Each cell type in the body contains a distinctive set
Portions of the cell membrane become part of the endo-
of cell surface and cytoplasmic receptor proteins that enable
cytotic vesicles or vacuoles during endocytosis; during exo-
it to respond to a complementary set of signaling molecules
cytosis, membrane is returned to the cell surface. This process
in a specific, programmed way. Cells bearing receptors for a
of membrane movement and recycling is called membrane
specific ligand are referred to as target cells for that molecule.
trafficking (Figure 2–7a). Trafficking of membrane com-
The routes of signal molecules from source to target provide
ponents occurs continuously in most cells and is not only
one way to categorize the signaling process:
crucial for maintaining the cell but also for physiologically
important processes such as reducing blood lipid levels. ■âŒ€ In endocrine signaling, the signal molecules (here
In many cells subpopulations of vacuoles and tubules called hormones) are carried in the blood from their
within the endosomal compartment accumulate small vesicles sources to target cells throughout the body.
within their lumens by further invaginations of their limiting ■âŒ€ In paracrine signaling, the chemical ligand diffuses in
membranes, becoming multivesicular bodies. While multi- extracellular fluid but is rapidly metabolized so that its
vesicular bodies may merge with lysosomes for selective deg- effect is only local on target cells near its source.
radation of their content, this organelle may also fuse with the ■âŒ€ In synaptic signaling, a special kind of paracrine inter-
plasma membrane and release the intralumenal vesicles out- action, neurotransmitters act on adjacent cells through
side the cell. The small (<120 nm diameter) vesicles released special contact areas called synapses (see Chapter 9).
â•‡
TABLE 2–2 Mechanisms of transport across the plasma membrane.
Process Type of Movement Example
PASSIVE PROCESSES Movement of substances down a concentration gradient due to the kinetic energy of the substance;
no expenditure of cellular energy is required; continues until equilibrium is reached (if unopposed)
Simple diffusion Unassisted net movement of small, nonpolar Exchange of oxygen and carbon dioxide
substances down their concentration gradient across a between blood and body tissues
selectively permeable membrane
Facilitated diffusion Movement of ions and small, polar molecules
down their concentration gradient; assisted across
a selectively permeable membrane by a transport
protein
â•…Channel-mediated Movement of ion down its concentration gradient Na+ moves through Na+ channel into cell
through a protein channel
â•…Carrier-mediated Movement of small, polar molecule down its Transport of glucose into cells by glucose
concentration gradient by a carrier protein carrier
Osmosis Diffusion of water across a selectively permeable Solutes in blood in systemic capillaries
membrane; direction is determined by relative solute “pulls” fluid from interstitial space back into
concentrations; continues until equilibrium the blood
is reached
ACTIVE PROCESSES Movement of substances requires expenditure of cellular energy
Active transport Transport of ions or small molecules across the
membrane against a concentration gradient by
transmembrane protein pumps
â•…Primary Movement of substance up its concentration gradient; Ca2+ pumps transport Ca2+ out of the cell
powered directly by ATP Na+/K+ pump moves Na+ out of cell and K+
into cell
â•…Secondary Movement of a substance up its concentration
gradient is powered by harnessing the movement of
a second substance (eg, Na+) down its concentration
gradient
â•…Symport Movement of substance up its concentration gradient Na+/glucose transport
in the same direction as Na+
â•…Antiport Movement of substance up its concentration gradient Na+/H+ transport
in the opposite direction from Na+
Vesicular transport Vesicle formed or lost as material is brought into a cell
or released from a cell
â•…Exocytosis Bulk movement of substance out of the cell by fusion Release of neurotransmitter by nerve cells
of secretory vesicles with the plasma membrane
â•…Endocytosis Bulk movement of substances into the cell by vesicles
forming at the plasma membrane
â•…Phagocytosis Type of endocytosis in which vesicles are formed as White blood cell engulfing a bacterium
particulate materials external to the cell are engulfed
by pseudopodia
â•…Pinocytosis Type of endocytosis in which vesicles are formed as Formation of small vesicles in capillary wall
interstital fluid is taken up by the cell to move substances
â•…Receptor-mediated endocytosis Type of endocytosis in which plasma membrane Uptake of cholesterol into cells
receptors first bind specific substances; receptor and
bound substance then taken up by the cell
FIGURE 2–6â•‡ Three major forms of endocytosis.
C H A P T E R
Extracellular fluid
Pseudopodia
Particle
2
Plasma
Plasma Vesicle membrane

membrane
Vacuole
Cytoplasm
a Phagocytosis
b Pinocytosis
Receptors
and then internalize this material into a cytoplasmic vacuole or

Plasma phagosome.
membrane (b) In pinocytosis the cell membrane forms similar folds or
invaginates (dimples inward) to create a pit containing a drop of
extracellular fluid. The pit pinches off inside the cell when the cell
membrane fuses and forms a pinocytotic vesicle containing the
Cytoplasmic
vesicle fluid.
(c) Receptor-mediated endocytosis includes membrane pro-
teins called receptors that bind specific molecules (ligands).
c Receptor-mediated endocytosis When many such receptors are bound by their ligands, they
aggregate in one membrane region, which then invaginates and
pinches off to create a vesicle or endosome containing both the
There are three general types of endocytosis:
receptors and the bound ligands.
(a) Phagocytosis involves the extension from the cell of surface
folds or pseudopodia which engulf particles such as bacteria,
■âŒ€ In autocrine signaling, signals bind receptors on the

same cells that produced the messenger molecule. › â•ºâ•º MEDICAL APPLICATION
■âŒ€ In juxtacrine signaling, important in early embryonic Many diseases are caused by defective receptors. For
tissue interactions, the signaling molecules are cell mem- example, pseudohypoparathyroidism and one type of
brane–bound proteins which bind surface receptors of dwarfism are caused by nonfunctioning parathyroid and
the target cell when the two cells make direct physical growth hormone receptors, respectively. In these two con-
contact. ditions the glands produce the respective hormones, but
the target cells cannot respond because they lack normal
Receptors for hydrophilic signaling molecules, includ-
receptors.
ing polypeptide hormones and neurotransmitters, are usu-
ally transmembrane proteins in the plasmalemma of target
cells. Three important functional classes of such receptors are
Ligands binding such receptors in a cell membrane can
shown in Figure 2–8:
be considered first messengers, beginning a process of signal
■âŒ€ Channel-linked receptors open associated channels transduction by activating a series of intermediary enzymes
upon ligand binding to promote transfer of molecules or downstream to produce changes in the cytoplasm, the
ions across the membrane. nucleus, or both. Channel-mediated ion influx or activation of
■âŒ€ Enzymatic receptors, in which ligand binding induces kinases can activate various cytoplasmic proteins, amplifying
catalytic activity in associated peripheral proteins. the signal. Activated G proteins target ion channels or other
■âŒ€ G protein–coupled receptors upon ligand binding membrane-bound effectors that also propagate the signal
stimulate associated G proteins which then bind the further into the cell (Figure 2–8). One such effector protein
guanine nucleotide GTP and are released to activate is the enzyme adenyl cyclase which generates large quanti-
other cytoplasmic proteins. ties of second messenger molecules, such as cyclic adenosine
FIGURE 2–7â•‡ Receptor-mediated endocytosis involves regulated membrane trafficking.
Receptor
Ligand ligand Clathrin
complexes coat
Receptors
Coated pit
Apical
domain Dynamin
of cell Adaptor
membrane protein Clathrin
Coat
proteins Coated CP CP
recycled vesicle
CV
Receptor
recycling
Early
endosome
Late
endosome
Transcytosis b
Lysosomal
degradation
Basolateral domain
of cell membrane
Major steps during and after endocytosis are indicated diagram- ■âŒ€ Receptors and ligands may be carried to late endosomes and
matically in part a. Ligands bind with high affinity to specific then to lysosomes for degradation.
surface receptors, which then associate with specific cytoplasmic ■âŒ€ Ligands may be released from the receptors and the empty
proteins, including clathrin and adaptor proteins, and aggregate receptors sequestered into recycling endosomes and
in membrane regions to form coated pits. Clathrin facilitates returned to the cell surface for reuse.
invagination of the pits, and another peripheral membrane pro- ■âŒ€ Other endosomal vesicles containing ligands may move
tein, dynamin, forms constricting loops around the developing to and fuse with another cell surface, where the ligands
neck of the pit, causing the invagination to pinch off as a coated are released again outside the cell in the process of
vesicle. The clathrin lattice of coated pits (CP) and vesicles (CV) is transcytosis.
shown ultrastructurally in part b.
(Figure 2–7b, used with permission from Dr John Heuser,
Internalized vesicles lose their clathrin coats, which are
Department of Cell Biology and Physiology, Washington University
recycled, and fuse with other endosomes that comprise the endo-
School of Medicine, St. Louis, MO.)
somal compartment. Ligands may have different fates within the
endosomal compartment:
monophosphate (cAMP). Other second messengers include carrier proteins in the plasma for transport through the body.
1,2-diacylglycerol (DAG), and inositol 1,4,5-triphosphate (IP3). Such hormones are lipophilic and pass by diffusion through
The ionic changes or second messengers amplify the first signal cell membranes, binding to specific cytoplasmic receptor
and trigger a cascade of enzymatic activity, usually including proteins in target cells. With many steroid hormones, recep-
kinases, leading to changes in gene expression or cell behavior. tor binding activates that receptor, enabling the complex to
Second messengers may diffuse through the cytoplasm or be move into the nucleus and bind with high affinity to specific
retained locally by scaffold proteins for more focused amplifica- DNA sequences. This generally increases the level of tran-
tion of activity. scription of those genes. Each steroid hormone is recognized
Low molecular weight hydrophobic signaling molecules, by a different member of a family of homologous receptor
such as steroids and thyroid hormones, bind reversibly to proteins.
Cytoplasmic Organelles 27
FIGURE 2–8â•‡ Major types of membrane receptors.
C H A P T E R
Channel open
Ions
Ligand g
Ligand
2
Channel
The Cytoplasmâ•‡ ■â•‡ Cytoplasmic Organelles

closed
Inactive protein Active protein

kinase enzyme kinase enzyme
phosphorylates
other enzymes.
Ions
Phosphate Enzyme turned
on or turned off
a Channel-linked receptors b Enzymatic receptors
1 A ligand binds to a
receptor, causing a Ions
conformational
connfor
format
ma ion
mational
al cha
change
nge
Ligand to
o activate
ac
activa receptor.
tivate recep
cep
ceptor
e tor
tor. Effector protein (eg, ion channel)
2 G protein binds to activated Inactive protein

receptor. kinase enzyme 5 Active protein
kinase enzyme
phosphorylates other
Second enzymes
messenger
Activated
Ac iva
Act ivated
ted
d
prot
ro
otein
ote
ei
G protein
GTP 3 GTP binds to G protein Phosphate
4 The activated effector protein
causing G-protein activation. makes secondary messenger
Activated G protein leaves the Effector protein
available within the cell, which
receptor. It attaches to and (eg, enzyme)
leads to protein kinase Enzyme turned
activates an effector protein. enzyme activation. on or turned off
(an ion channel or an enzyme).
c G-protein–coupled receptors
Protein and most small ligands are hydrophilic molecules that bind phosphorylate (and usually activate) other proteins upon ligand
transmembrane protein receptors to initiate changes in the target cell. binding. (c) G-protein–coupled receptors bind ligand, changing
(a) Channel-linked receptors bind ligands such as neurotrans- the conformation of its G-protein subunit, allowing it to bind GTP,
mitters and open to allow influx of specific ions. (b) Enzymatic and activating and releasing this protein to in turn activate other
receptors are usually protein kinases that are activated to proteins such as ion channels and adenyl cyclase.
›â•ºCYTOPLASMIC ORGANELLES energy. Oxygen, CO2, electrolytic ions, low-molecular-weight

substrates, metabolites, and waste products all diffuse through
Inside the cell membrane the fluid cytoplasm (or cytosol) cytoplasm, either freely or bound to proteins, entering or leav-
bathes metabolically active structures called organelles, ing organelles where they are used or produced.
which may be membranous (such as mitochondria) or non-
membranous protein complexes (such as ribosomes and pro-
teasomes). Most organelles are positioned in the cytoplasm by Ribosomes
movements along the polymers of the cytoskeleton, which Ribosomes are macromolecular machines, about 20 × 30 nm
also determines a cell’s shape and motility. in size, which assemble polypeptides from amino acids on
Cytosol also contains hundreds of enzymes, such as those molecules of transfer RNA (tRNA) in a sequence specified
of the glycolytic pathway, which produce building blocks for by mRNA. A functional ribosome has two subunits of differ-
larger molecules and break down small molecules to liberate ent sizes bound to a strand of mRNA. The core of the small
ribosomal subunit is a highly folded ribosomal RNA (rRNA) Proper folding of new proteins is guided by protein chaper-
chain associated with more than 30 unique proteins; the core ones. Denatured proteins or those that cannot be refolded prop-
of the large subunit has three other rRNA molecules and erly are conjugated to the protein ubiquitin that targets them for
nearly 50 other basic proteins. breakdown by proteasomes (discussed below). As indicated in
The rRNA molecules in the ribosomal subunits not only Figure 2–9, proteins synthesized for use within the cytosol (eg,
provide structural support but also position transfer RNAs glycolytic enzymes) or for import into the nucleus and certain
(tRNA) molecules bearing amino acids in the correct “reading other organelles are synthesized on polyribosomes existing as
frame” and catalyze the formation of the peptide bonds. The isolated cytoplasmic clusters. Polyribosomes attached to mem-
more peripheral proteins of the ribosome seem to function branes of the endoplasmic reticulum (ER) translate mRNAs
primarily to stabilize the catalytic RNA core. coding for membrane proteins of the ER, the Golgi apparatus,
These ribosomal proteins are themselves synthesized in or the cell membrane; enzymes to be stored in lysosomes; and
cytoplasmic ribosomes, but are then imported to the nucleus proteins to undergo exocytosis from secretory vesicles.
where they associate with newly synthesized rRNA. The ribo-
somal subunits thus formed then move from the nucleus to
the cytoplasm where they are reused many times, for transla- Endoplasmic Reticulum
tion of any mRNA strand. The cytoplasm of most cells contains a convoluted membranous
During protein synthesis many ribosomes typically bind network called the endoplasmic reticulum (ER). As shown in
the same strand of mRNA to form larger complexes called Figure 2–10 this network (reticulum) extends from the surface
polyribosomes, or polysomes (Figure 2–9). In stained of the nucleus throughout most of the cytoplasm and encloses
preparations of cells polyribosomes are intensely basophilic a series of intercommunicating channels called cisternae
because of the numerous phosphate groups of the constitu- (L. cisternae, reservoirs). With a membrane surface up to 30 times
ent RNA molecules that act as polyanions. Thus, cytoplasmic that of the plasma membrane, the ER is a major site for vital
regions that stain intensely with hematoxylin and basic dyes, cellular activities, including biosynthesis of proteins and lipids.
such as methylene and toluidine blue, indicate sites of active Numerous polyribosomes attached to the membrane in some
protein synthesis. regions of ER allow two types of ER to be distinguished.
FIGURE 2–9â•‡ Polyribosomes: free or bound to the endoplasmic reticulum.
3′
FREE POLYRIBOSOMES 5′ ER-BOUND POLYRIBOSOMES 3′
5′
mRNA
Ribosome
Cisterna of
rough ER
Misfolded &
Proteins of cytosol denatured proteins
& cytoskeleton
Conjugated to ubiquitin Golgi apparatus processing & sorting
Specific proteins imported to Secretory vesicles Lysosomes
Mitochondria Peroxisomes Nucleus Proteasome Proteins secreted Proteins of cell

Protein degradation from cell membrane
Free polyribosomes (not attached to the endoplasmic reticulum, polysomes attached to the membranes of ER. The proteins pro-
or ER) synthesize cytosolic and cytoskeletal proteins and proteins duced by these ribosomes are segregated during translation into
for import into the nucleus, mitochondria, and peroxisomes. the interior of the ER’s membrane cisternae.
Proteins that are to be incorporated into membranes, stored In both pathways misfolded proteins are conjugated to ubiqui-
in lysosomes, or eventually secreted from the cell are made on tin and targeted for proteasomal degradation.
FIGURE 2–10â•‡ Rough and smooth endoplasmic reticulum.
C H A P T E R
Nucleus
2
Cisternae
Ribosomes
a c
Ribosomes Rough ER
Smooth ER
Functions of Endoplasmic Reticulum
1. Synthesis: Provides a place for chemical reactions

a. Smooth ER is the site of lipid synthesis and carbohydrate metabolism
b. Rough ER synthesizes proteins for secretion, incorporation into the
plasma, membrane, and as enzymes within lysosomes
2. Transport: Moves molecules through cisternal space from one part of
the cell to another, sequestered away from the cytoplasm
3. Storage: Stores newly synthesized molecules
4. Detoxification: Smooth ER detoxifies both drugs and alcohol
(a) The endoplasmic reticulum is an anastomosing network of The interconnected membranous cisternae of RER are flattened,
intercommunicating channels or cisternae formed by a continu- while those of SER are frequently tubular. (14,000X)
ous membrane, with some regions that bear polysomes appearing (c) In a very thin cultured endothelial cell, both ER (green) and
rough and other regions appearing smooth. While RER is the site mitochondria (orange) can be visualized with vital fluorescent
for synthesis of most membrane-bound proteins, three diverse dyes that are sequestered specifically into those organelles. This
activities are associated with smooth ER: (1) lipid biosynthesis, staining method with intact cells clearly reveals the continuous,
(2) detoxification of potentially harmful compounds, and lacelike ER present in all regions of the cytoplasm.
(3) sequestration of Ca++ ions. Specific cell types with well-developed (Figure 2–10c, © 2015 Thermo Fisher Scientific, Inc. Used under
SER are usually specialized for one of these functions. permission.)
(b) By TEM cisternae of RER appear separated, but they actually
form a continuous channel or compartment in the cytoplasm.
Rough Endoplasmic Reticulum and proteins to be secreted by exocytosis. Production here

Rough endoplasmic reticulum (RER) is prominent in cells includes the initial (core) glycosylation of glycoproteins, cer-
specialized for protein secretion, such as pancreatic acinar tain other posttranslational modifications of newly formed
cells (making digestive enzymes), fibroblasts (collagen), and polypeptides, and the assembly of multichain proteins. These
plasma cells (immunoglobulins). The RER consists of saclike activities are mediated by resident enzymes of the RER and
as well as parallel stacks of flattened cisternae (Figure 2–10), by protein complexes that act as chaperones guiding the fold-
each limited by membranes that are continuous with the outer ing of nascent proteins, inhibiting aggregation, and generally
membrane of the nuclear envelope. The presence of polyribo- monitoring protein quality within the ER.
somes on the cytosolic surface of the RER confers basophilic Protein synthesis begins on polyribosomes in the cytosol.
staining properties on this organelle when viewed with the The 5′ ends of mRNAs for proteins destined to be segregated
light microscope. in the ER encode an N-terminal signal sequence of 15-40
The major function of RER is production of membrane- amino acids that includes a series of six or more hydropho-
associated proteins, proteins of many membranous organelles, bic residues. As shown in Figure 2–11, the newly translated
FIGURE 2–11â•‡ Movement of polypeptides into the RER.
mRNA tRNA
5′ Signal sequence
SRP binds to SRP is is removed from
SRP receptor liberated 3′
growing polypeptide
New polypeptide
with initial
signal peptide SRP bound to
signal peptide
SRP Ribosome receptor

Signal recognition receptor and protein Signal Growing
particle (SRP) translocator complex pepsidase polypeptide
Completed
RER membrane RER cisterna polypeptide
The newly translated amino terminus of a protein to be incorpo- ribosomal subunit, more firmly attaching the ribosome to the ER.
rated into membranes or sequestered into vesicles contains 15-40 The hydrophobic signal peptide is translocated through a protein
amino acids that include a specific sequence of hydrophobic pore (translocon) in the ER membrane, and the SRP is freed for
residues comprising the signal sequence or signal peptide. This reuse. The signal peptide is removed from the growing protein by
sequence is bound by a signal-recognition particle (SRP), which a peptidase and translocation of the growing polypeptide contin-
then recognizes and binds a receptor on the ER. Another recep- ues until it is completely segregated into the ER cisterna.
tor in the ER membrane binds a structural protein of the large
signal sequence is bound by a protein complex called the differences in the destinations of their major protein products
signal-recognition particle (SRP), which inhibits further and how these differences determine a cell’s histologic features.
polypeptide elongation. The SRP-ribosome-nascent peptide
complex binds to SRP receptors on the ER membrane. SRP
then releases the signal sequence, allowing translation to › â•ºâ•º MEDICAL APPLICATION
continue with the nascent polypeptide chain transferred to a Quality control during protein production in the RER and
translocator complex (also called a translocon) through the properly functioning ERAD to dispose of defective proteins
ER membrane (Figure 2–11). Inside the lumen of the RER, are extremely important and several inherited diseases result
the signal sequence is removed by an enzyme, signal pepti- from malfunctions in this system. For example, in some forms
dase. With the ribosome docked at the ER surface, translation of osteogenesis imperfecta bone cells synthesize and secrete
continues with the growing polypeptide pushing itself while defective procollagen molecules which cannot assemble
chaperones and other proteins serve to “pull” the nascent poly- properly and produce very weak bone tissue.
peptide through the translocator complex. Upon release from
the ribosome, posttranslational modifications and proper Smooth Endoplasmic Reticulum
folding of the polypeptide continue.
Regions of ER that lack bound polyribosomes make up the
RER has a highly regulated system to prevent nonfunc-
tional proteins being forwarded to the pathway for secretion smooth endoplasmic reticulum (SER), which is continuous
or to other organelles. New proteins that cannot be folded with RER but frequently less abundant (Figure 2–10). Lack-
or assembled properly by chaperones undergo ER-associated ing polyribosomes, SER is not basophilic and is best seen with
degradation (ERAD), in which unsalvageable proteins are the TEM. Unlike the cisternae of RER, SER cisternae are more
translocated back into the cytosol, conjugated to ubiquitin, tubular or saclike, with interconnected channels of various
and then degraded by proteasomes. shapes and sizes rather than stacks of flattened cisternae.
As mentioned, proteins synthesized in the RER can have SER has three main functions, which vary in importance
several destinations: intracellular storage (eg, in lysosomes in different cell types.
and specific granules of leukocytes), provisional storage in ■âŒ€ Enzymes in the SER perform synthesis of phospholipids
cytoplasmic vesicles prior to exocytosis (eg, in the pancreas, and steroids, major constituents of cellular membranes.
some endocrine cells), and as integral membrane proteins. These lipids are then transferred from the SER to
Diagrams in Figure 2–12 show a few cell types with distinct other membranes by lateral diffusion into adjacent
FIGURE 2–12â•‡ Protein localization and cell morphology.
2 C H A P T E R
(a) Erythroblast (b) Eosinophilic leukocyte (c) Plasma cell (d) Pancreatic acinar cell
The ultrastructure and general histologic appearance of a cell are (c) Cells with extensive RER and a well-developed Golgi apparatus
determined by the nature of the most prominent proteins the cell show few secretory granules because the proteins undergo exocytosis
is making. immediately after Golgi processing is complete. Many cells, especially
(a) Cells that make few or no proteins for secretion have very little those of epithelia, are polarized, meaning that the distribution of RER
RER, with essentially all polyribosomes free in the cytoplasm. and secretory vesicles is different in various regions or poles of the cell.
(b) Cells that synthesize, segregate, and store various proteins in (d) Epithelial cells specialized for secretion have distinct polarity,
specific secretory granules or vesicles always have RER, a Golgi with RER abundant at their basal ends and mature secretory gran-
apparatus, and a supply of granules containing the proteins ready ules at the apical poles undergoing exocytosis into an enclosed
to be secreted. extracellular compartment, the lumen of a gland.
membranes, by phospholipid transfer proteins, or by Golgi Apparatus

vesicles which detach from the SER for movement along
The dynamic organelle called the Golgi apparatus, or Golgi
the cytoskeleton and fusion with other membranous
complex, completes posttranslational modifications of pro-
organelles. In cells that secrete steroid hormones (eg,
teins produced in the RER and then packages and addresses
cells of the adrenal cortex), SER occupies a large portion
these proteins to their proper destinations. The organelle was
of the cytoplasm.
named after histologist Camillo Golgi who discovered it in
■âŒ€ Other SER enzymes, including those of the cyto-
1898. The Golgi apparatus consists of many smooth membra-
chrome P450 family, allow detoxification of potentially
nous saccules, some vesicular, others flattened, but all con-
harmful exogenous molecules such as alcohol, barbitu-
taining enzymes and proteins being processed (Figure 2–13).
rates, and other drugs. In liver cells these enzymes also
In most cells the small Golgi complexes are located near the
process endogenous molecules such as the components
nucleus.
of bile.
As shown in Figure 2–13, the Golgi apparatus has two dis-
■âŒ€ SER vesicles are also responsible for sequestration and
tinct functional sides or faces, formed by the complex traffic of
controlled release of Ca2+, which is part of the rapid
vesicles within cells. Material moves from the RER cisternae
response of cells to various stimuli. This function is par-
to the Golgi apparatus in small, membrane-enclosed carriers
ticularly well developed in striated muscle cells, where
called transport vesicles that are transported along cytoskel-
the SER has an important role in the contraction process
etal polymers by motor proteins. The transport vesicles merge
and assumes a specialized form called the sarcoplasmic
with the Golgi-receiving region, or cis face. On the opposite
reticulum (see Chapter 10).
side of the Golgi network, at its shipping or trans face, larger
saccules or vacuoles accumulate, condense, and generate other
› â•ºâ•º MEDICAL APPLICATION vesicles that carry completed protein products to organelles
Jaundice denotes a yellowish discoloration of the skin and is
away from the Golgi (Figure 2–13).
caused by accumulation in extracellular fluid of bilirubin and
Formation of transport vesicles and secretory vesicles
other pigmented compounds, which are normally metabo-
is driven by assembly of various coat proteins (including
lized by SER enzymes in cells of the liver and excreted as bile.
clathrin), which also regulate vesicular traffic to, through,
A frequent cause of jaundice in newborn infants is an under-
and beyond the Golgi apparatus. Forward movement of ves-
developed state of SER in liver cells, with failure of bilirubin to
icles in the cis Golgi network of saccules is promoted by the
be converted to a form that can be readily excreted.
coat protein COP-II, while retrograde movements in that
region involve COP-I. Other membrane proteins important
FIGURE 2–13â•‡ Golgi apparatus.
TV Vacuole
shipping
region
CF Secretory
vesicles
SV
TF
SV
Transport
Transport
vesicle
vesicle
Lumen of cisterna
filled with secretory
TV product
ER
G
M
b c
The Golgi apparatus is a highly plastic, morphologically complex (b) Morphological aspects of the Golgi apparatus are revealed
system of membrane vesicles and cisternae in which proteins and more clearly by SEM, which shows a three-dimensional snapshot
other molecules made in the RER undergo further modification of the region between RER and the Golgi membrane compart-
and sorting into specific vesicles destined for different roles in the ments. Cells may have multiple Golgi apparatuses, each with the
cell. general organization shown here and typically situated near the
(a) TEM of the Golgi apparatus provided early evidence about cell nucleus. (X30,000)
how this organelle functions. To the left is a cisterna of RER and (c) The Golgi apparatus location can be clearly seen in intact cul-
close to it are many small vesicles at the cis face (CF), or receiv- tured cells processed by immunocytochemistry using an antibody
ing face, of the Golgi apparatus, merging with the first of several against golgin-97 to show the many complexes of Golgi vesicles
flattened Golgi cisternae. In the center are the characteristic flat- (green), all near the nucleus, against a background of microfila-
tened, curved, and stacked medial cisternae of the complex. Cyto- ments organized as stress fibers and stained with fluorescent
logical and molecular data suggest that other transport vesicles phalloidin (violet). Because of the abundance of lipids in its many
(TV) move proteins serially through the cisternae until at the trans membranes, the Golgi apparatus is difficult to visualize in typical
face (TF), or shipping region, larger condensing secretory vesicles paraffin-embedded, H&E-stained sections. In developing white
(SV) and other vacuoles emerge to carry the modified proteins. blood cells with active Golgi complexes, the organelle can some-
elsewhere in the cell. Formation and fusion of the vesicles through times be seen as a faint unstained juxtanuclear region (sometimes
the Golgi apparatus is controlled by specific membrane proteins. called a “Golgi ghost”) surrounded by basophilic cytoplasm.
(X30,000) Inset: A small region of a Golgi apparatus in a 1-μm (Figure 2–13b reproduced, with permission from Naguro T, Iino A.
section from a silver-stained cell, demonstrating abundant glyco- Prog Clin Biol Res. 1989;295:250; Figure 2–13c, © 2015 Thermo Fisher
proteins within cisternae. Scientific, Inc. Used under permission.)
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kauan kuin hän elää! Semmoinen on päätökseni! Ivan Fjodorovitš
hyväksyy menettelyni aivan täydellisesti.
Hän läähätti. Kenties hän olisi tahtonut lausua ajatuksensa paljon

arvokkaammin, taitavammin ja luonnollisemmin, mutta tuli tehneeksi
sen liian kiireesti ja liian avoimesti. Paljon tuli siihen nuorta
itsensähillitsemisen puutetta, paljon siinä johtui vain eilisestä
kiihtymyksestä, ylpeilyn tarpeesta, sen hän tunsi itse. Hänen
kasvonsa tulivat äkkiä omituisen synkiksi, silmien ilme tuli
epämiellyttäväksi. Aljoša huomasi heti kaiken tämän, ja hänen
sydämensä liikahti säälistä. Mutta samassa veli Ivan lisäsi:
— Minä lausuin vain ajatukseni, — sanoi hän. — Jonkun toisen

sanomana tämä kaikki olisi tehnyt masentuneen ja kiusaantuneen
vaikutuksen, mutta ei teidän sanomananne. Joku toinen olisi ollut
väärässä, mutta te olette oikeassa! Minä en tiedä, kuinka tämä olisi
perusteltava, mutta näen, että te olette mitä suurimmassa määrässä
vilpitön, ja siksi te olette oikeassa…
— Mutta tämähän on vain tällä hetkellä… Mutta mitä on tämä

hetki? Vain eilinen loukkaus, — sitä merkitsee tämä hetki! — ei
rouva Hohlakov malttanut olla äkkiä sanomatta. Ilmeisesti hän ei
ollut tahtonut sekaantua asiaan, mutta ei ollut voinut hillitä itseään ja
tuli yht'äkkiä lausuneeksi sangen oikean ajatuksen.
— Niinpä kyllä, — keskeytti Ivan jonkin verran kiihkeästi ja

nähtävästi vihaisena siitä, että hänet oli keskeytetty, — niin, mutta
jollekulle toiselle tämä hetki olisi vain eilinen vaikutelma ja vain yksi
hetki, kun taas sellaiselle luonteelle kuin Katerina Ivanovna tämä
hetki tulee koko elämän pituiseksi. Mikä toisille on vain lupaus, se on
hänelle ikuinen, raskas, kenties murheellinen velvollisuus, josta ei
voi luopua. Ja tämä täytetyn velvollisuuden tunne on ylläpitävä
häntä. Teidän elämänne, Katerina Ivanovna, kuluu nyt omien
tunteittenne, oman sankarillisen tekonne ja kärsimyksiä tuottavassa
oman murheenne katselemisessa, mutta myöhemmin tuo kärsimys
lieventyy ja elämänne muuttuu silloin kerta kaikkiaan täytetyn lujan ja
ylpeän ajatuksen suloiseksi katselemiseksi, ajatuksen, joka
todellakin on omalla tavallaan ylpeä ja joka tapauksessa
epätoivoinen, mutta jossa te olette saavuttanut voiton, ja tämä
tietoisuus tuottaa teille lopulta mitä täydellisimmän hyvityksen ja saa
teidät sovintoon kaiken muun kanssa…
Hän puhui tämän päättävästi, jollakin tavoin häijysti, ilmeisesti

tahallaan ja kenties tahtomatta salatakaan tarkoitustaan, nimittäin
puhua tahallansa ja ivallisesti.
— Voi hyvä Jumala, kuinka toisin tuo kaikki on! — huudahti taas
rouva
Hohlakov.
— Aleksei Fjodorovitš, sanokaa te! Minua kiusaa halu saada

tietää, mitä te minulle sanotte! — huudahti Katerina Ivanovna, ja
kyynelet tulvahtivat äkkiä hänen silmistään. Aljoša nousi sohvalta.
— Ei tämä ole mitään, ei mitään! — jatkoi Katerina Ivanovna

itkien. — Tämä johtuu tilapäisestä heikkoudesta, viime yöstä, mutta
kahden sellaisen ystävän rinnalla kuin te ja veljenne minä tunnen,
että minulla vielä on voimia… koska tiedän… että te kumpikaan ette
koskaan jätä minua.
— Valitettavasti minun täytyy kenties jo huomenna lähteä

Moskovaan ja jättää teidät pitkäksi aikaa… Eikä tätä, paha kyllä, voi
muuttaa… — lausui äkkiä Ivan Fjodorovitš.
— Huomenna Moskovaan! — sanoi Katerina Ivanovna, ja hänen
kasvonsa vääntyivät yht'äkkiä. — Mutta… mutta hyvä Jumala,
kuinka onnellista se on! — huudahti hän äänellä, joka oli
silmänräpäyksessä kokonaan muuttunut. Silmänräpäyksessä olivat
myös kyynelet kadonneet hänen silmistään aivan jäljettömiin.
Nimenomaan juuri silmänräpäyksessä oli hänessä tapahtunut
ihmeellinen muutos, joka suuresti kummastutti Aljošaa: äskeisen
loukatun tyttöraukan sijaan, joka oli itkenyt jonkinmoisen
tunteenpurkauksen vallassa, oli äkkiä tullut nainen, joka täydellisesti
hillitsi itsensä, vieläpä oli erittäin tyytyväinen johonkin, aivan kuin olisi
äkkiä ilostunut jostakin.
— Oi, ei se ole onnellista, että minä menetän teidät, ei tietenkään,

— korjasi hän tavallaan äskeistä puhettaan ja hymyili kohteliasta
hymyä, — sellainen ystävä kuin te ei voi sitä luulla. Olen päinvastoin
onneton menettäessäni teidät (hän syöksyi äkkiä rajusti Ivan
Fjodorovitšin luo, tarttui hänen molempiin käsiinsä ja puristi niitä
kiihkeästi). Mutta onnellista on se, että te itse persoonallisesti voitte
nyt kertoa Moskovassa tädille ja Agafjalle koko minun asemani, koko
minun nykyisen kauhean kohtaloni, täysin avomielisesti Agafjalle ja
säästäen rakasta tätiä, niinkuin itse sen kyllä ymmärrätte tehdä. Ette
voi kuvitellakaan, kuinka onneton olin eilen ja tänä aamuna, kun en
tietänyt, miten kirjoittaisin heille tämän kauhean kirjeen… sillä
kirjeessä ei tätä voi mitenkään esittää… Mutta nyt minun on helppo
kirjoittaa, koska te olette siellä heidän luonaan ja selitätte kaiken. Oi,
kuinka iloinen olen! Mutta iloitsen vain tästä, uskokaa taaskin minua.
Teitä itseänne ei minulle tietysti mikään voi korvata… Riennän heti
kirjoittamaan kirjeen, — lopetti hän äkkiä ja astui jo askelen
lähteäkseen huoneesta.
— Entä Aljoša? Entä Aleksei Fjodorovitšin mielipide, joka teidän
niin välttämättömästi piti saada tietää? — huudahti rouva Hohlakov.
Hänen äänessään oli pisteliäs ja vihainen sävy.
— Minä en ole unohtanut sitä, — pysähtyi Katerina Ivanovna

äkkiä, — mutta miksi te olette minua kohtaan niin vihamielinen
tämmöisellä hetkellä, Katerina Osipovna? — lausui hän katkerasti ja
kiihkeästi soimaten. — Mitä minä sanoin, sen myös vahvistan
todeksi. Minun on välttämätöntä saada kuulla hänen mielipiteensä,
vieläpä enemmänkin: minä tarvitsen hänen ratkaisuaan! Mitä hän
sanoo, niin tapahtuu — niin suuressa määrin minä päinvastoin
himoitsen kuulla sanojanne, Aleksei Fjodorovitš… Mutta mikä teitä
vaivaa?
— Minä en ole koskaan luullut, minä en voi sitä kuvitella! —

huudahti äkkiä Aljoša surullisesti.
— Mitä, mitä?
— Hän menee Moskovaan, ja te huudahditte olevanne iloinen —

sen te teitte tahallanne! Sitten te heti aloitte selitellä, että ette ole
siitä iloinen, vaan että teistä päinvastoin on ikävä, kun… menetätte
ystävän, — mutta senkin te näyttelitte tahallanne… aivan kuin
teatterissa, näyttelitte kuin komediaa!…
— Teatterissa? Kuinka… Mitä tämä on? — huudahti Katerina

Ivanovna suuresti hämmästyen ja kiivastuneena sekä kulmiaan
rypistäen.
— Vakuuttakaapa hänelle miten paljon tahansa, että te kaipaatte

häntä ystävänä, niin te kuitenkin väitätte hänelle päin silmiä, että on
onni, kun hän matkustaa pois… — lausui Aljoša ikäänkuin
menehtyneenä. Hän seisoi pöydän ääressä eikä istuutunut.
— Mitä te tarkoitatte, minä en ymmärrä…
— Enhän minä itsekään tiedä… Minulle on äkkiä ikäänkuin

valjennut… Minä tiedän puhuvani pahasti, mutta sanon kuitenkin
kaiken, — jatkoi Aljoša yhtä värisevällä ja katkonaisella äänellä. —
Minulle on valjennut semmoista, että te kenties ette ollenkaan
rakasta veli Dmitriä… alusta alkaen ette ole rakastanut… Ja kenties
myös Dmitri ei teitä ollenkaan rakasta… alusta pitäen… vaan
ainoastaan kunnioittaa… Minä en todellakaan tiedä, kuinka nyt
uskallan tästä kaikesta puhua, mutta täytyyhän jonkun sanoa
totuus… sillä kukaan ei täällä tahdo sanoa totuutta…
— Mitä totuutta? — huudahti Katerina Ivanovna, ja hänen

äänessään oli hysteerinen kaiku.
— Tätä, — mutisi Aljoša aivan kuin olisi puusta pudonnut, —

kutsukaa heti tänne Dmitri — minä etsin hänet käsiin — ja tulkoon
hän tänne ja ottakoon teitä kädestä, ottakoon sitten kädestä veli
Ivania ja yhdistäköön teidän kätenne. Sillä te kiusaatte Ivania vain
sen tähden, että rakastatte häntä… ja kiusaatte sen tähden, että
rakastatte Dmitriä jonkin puuskan vuoksi… rakastatte epätodesti…
koska olette itsellenne niin vakuuttanut…
Aljošan puhe katkesi ja hän vaikeni.
— Te… te… te… olette pieni hassu, sitä te olette! — sanoi äkkiä
Katerina Ivanovna kasvot kalvenneina ja huulet vihasta vääntyneinä.
Ivan Fjodorovitš alkoi nauraa ja nousi paikaltaan. Hänellä oli hattu
kädessään.
— Sinä olet erehtynyt, hyvä Aljošani, — lausui hän, ja hänen
kasvoissaan oli sellainen ilme, jommoista Aljoša ei ollut niissä vielä
koskaan nähnyt, — jonkinmoista nuorekasta vilpittömyyttä ja
voimakasta, hillittömän avomielistä tunnetta, — ei koskaan ole
Katerina Ivanovna rakastanut minua! Hän on tietänyt kaiken aikaa,
että minä rakastan häntä, vaikka minä en koskaan ole puhunut
hänelle sanaakaan rakkaudestani, hän on — tietänyt, mutta ei ole
rakastanut minua. En myöskään ole ollut hänen ystävänsä
kertaakaan, ainoatakaan päivää: ylpeä nainen ei ole tarvinnut
ystävyyttäni. Hän piti minua läheisyydessään voidakseen yhtä mittaa
kostaa. Hän kosti minulle ja minussa kaikki loukkaukset, joita hän
alituisesti ja joka hetki koko tänä aikana on saanut kärsiä Dmitrin
puolelta, loukkaukset heidän ensimmäisestä kohtaamisestaan asti…
Sillä heidän aivan ensimmäinenkin kohtauksensa on jäänyt hänen
sydämeensä loukkaukseksi. Sellainen on hänen sydämensä! Minä
en koko aikana ole muuta tehnytkään kuin kuullut hänen
rakkaudestaan Dmitriin. Minä lähden nyt matkaan, mutta tietäkää,
Katerina Ivanovna, että te todellisuudessa rakastatte ainoastaan
häntä. Ja kuta enemmän hän loukkaa, sitä enemmän ja yhä
enemmän. Se juuri on teidän puuskanne. Te rakastatte häntä
semmoisena kuin hän on, rakastatte häntä teitä loukkaavana. Jos
hän tekisi parannuksen, niin te heti hylkäisitte hänet ja lakkaisitte
kokonaan rakastamasta. Mutta hän on teille tarpeellinen, jotta
voisitte katsoa lakkaamatta sankarillista uskollisuuttanne ja moittia
häntä uskottomuudesta. Ja kaikki tämä johtuu teidän ylpeydestänne.
Oi, tässä on paljon alentamista ja alentumista, mutta kaikki johtuu
ylpeydestä… Minä olen liian nuori ja olen rakastanut teitä liian
voimakkaasti. Minä tiedän, että minun ei pitäisi puhua teille tätä, että
olisi arvokkaampaa yksinkertaisesti poistua luotanne, ja se olisi
teillekin vähemmän loukkaavaa. Mutta minähän matkustan kauaksi
enkä palaa koskaan. Tämähän on ero iäksi… Minä en tahdo istua
puuskan ja mullerruksen vieressä… Muuten en osaa enää puhua,
olen sanonut kaiken… Hyvästi, Katerina Ivanovna, te ette saa olla
vihainen minulle, sillä minä olen saanut sata kertaa kovemman
rangaistuksen kuin te: se on jo minulle rangaistus, että en koskaan
saa nähdä teitä. Hyvästi. Minä en tarvitse kättänne. Te olette liian
tietoisesti minua kiusannut, jotta voisin tällä hetkellä antaa teille
anteeksi. Myöhemmin annan anteeksi, mutta nyt ei tarvita kättänne.
»Den Dank, Dame, begehr ich nicht!» — lisäsi hän vääristäen
suunsa hymyyn (osoittaen, muuten aivan odottamattomasti, että
hänkin pystyi lukemaan Schilleriä niin paljon, että osasi ulkoakin,
mitä Aljoša aikaisemmin ei olisi uskonut). Hän poistui huoneesta
sanomatta jäähyväisiä edes emännälle, rouva Hohlakoville. Aljoša löi
käsiään yhteen.
— Ivan, — hän huudahti hänen jälkeensä aivan kuin pyörällä

päästään, — tule takaisin, Ivan! Ei, ei, hän ei palaa nyt missään
tapauksessa! — huudahti hän murheellisena ja aivan kuin hänelle
taas olisi jotakin valjennut, — mutta se on minun, minun syyni, minä
aloitin! Ivan puhui häijysti, pahasti, epäoikeudenmukaisesti ja
ilkeästi… Hänen täytyy taas tulla tänne, palata, palata… — Aljoša
huudahteli kuin vähämielinen.
Katerina Ivanovna poistui äkkiä toiseen huoneeseen.
— Te ette ole tehnyt mitään, te olette toiminut ihanasti kuin enkeli,

— kuiskasi rouva Hohlakov nopeasti ja innostuneesti murheelliselle
Aljošalle. — Minä teen kaiken voitavani, että Ivan Fjodorovitš ei
matkustaisi pois…
Hänen kasvonsa loistivat ilosta Aljošan suureksi harmiksi, mutta

Katerina Ivanovna tuli yht'äkkiä takaisin. Hänellä oli kädessään kaksi
sadan ruplan seteliä.
— Minulla on teille eräs suuri pyyntö, Aleksei Fjodorovitš, — alkoi

hän kääntyen suoraan Aljošan puoleen ja näennäisesti rauhallisella
ja tyynellä äänellä, aivan kuin äsken tosiaankaan ei olisi mitään
tapahtunut. — Viikko — niin, luullakseni se oli viikko sitten — Dmitri
Fjodorovitš teki kiivastuneena erään sangen epäoikeutetun teon,
hyvin sopimattoman teon. Täällä on eräs huono paikka, muudan
ravintola. Siellä hän kohtasi erään virasta eronneen upseerin, tuon
alikapteenin, jota isänne on käyttänyt joihinkin asioihinsa.
Suuttuneena jostakin tähän alikapteeniin Dmitri Fjodorovitš tarttui
häntä partaan ja veti hänet kaikkien nähden tällä loukkaavalla tavalla
kadulle vedellen häntä vielä kadulla pitkän aikaa, ja kerrotaan tämän
alikapteenin pienen pojan, joka on oppilaana täkäläisessä koulussa
ja joka on vielä aivan lapsi, juosseen kaiken aikaa vieressä ääneen
itkien ja pyytäneen isänsä puolesta ja pyydelleen kaikkia muitakin
puolustamaan isää, mutta kaikki olivat vain nauraneet. Suokaa
anteeksi, Aleksei Fjodorovitš, mutta en voi olla paheksuen
muistelematta tätä häpeällistä tekoa… yhtä niistä teoista, jommoisiin
vain Dmitri Fjodorovitš voi ryhtyä vihapäissään… ja intohimojensa
vallassa! En osaa edes kertoa sitä, en kykene… Sekaannun
sanoissani. Olen ottanut selkoa tuosta pahoinpidellystä ja kuullut,
että hän on hyvin köyhä mies. Hänen sukunimensä on Snegirev.
Hän on tehnyt jonkin virkarikoksen, hänet on erotettu virasta, en
osaa kertoa sitä teille, ja nyt hän on perheensä kanssa, onnettoman
perheensä kanssa, johon kuuluu sairaita lapsia ja sairas vaimo,
mielenvikainen luullakseni, joutunut hirveään kurjuuteen. Hän on jo
kauan asunut kaupungissamme, hänellä on joitakin hommia, hän on
ollut jossakin kirjurina, mutta nyt hänelle on yht'äkkiä lakattu
maksamasta. Olen luonut silmäni teihin… toisin sanoen ajattelin, —
minä en tiedä, sanani sotkeutuvat, — nähkääs, minä olin aikonut
pyytää teitä, Aleksei Fjodorovitš, — hyvä Aleksei Fjodorovitši,
käymään hänen luonaan, jollakin tekosyyllä menemään heille,
nimittäin tuon alikapteenin luo, — voi hyvä Jumala, miten minä
sotken! — ja hienotunteisesti, varovasti, — juuri sillä tavoin kuin vain
te osaatte tehdä (Aljoša punastui äkkiä) — koettamaan saada
annetuksi hänelle avustus, nämä kaksisataa ruplaa. Hän ottaa
varmasti vastaan… toisin, sanoen hänet pitää suostuttaa ottamaan
vastaan… Tai ei, kuinka sanoisin? Katsokaahan, tämä ei ole mitään
sopijaisia hänelle, jotta hän ei nostaisi kannetta (sillä hän lienee
aikonut nostaa kanteen), vaan ainoastaan sääliä, auttamishalua
minun puoleltani, Dmitri Fjodorovitšin morsiamelta eikä häneltä
itseltään… Sanalla sanoen te osaatte kyllä… Menisin itse, mutta te
osaatte toimittaa sen paljon paremmin kuin minä. Hän asuu
Ozernaja-kadun varrella, pikkuporvarinleski Kalmykovin talossa…
Jumalan tähden, Aleksei Fjodorovitš, tehkää minulle tämä, minä…
minä olen nyt jonkin verran… väsynyt. Näkemiin…
Hän pyörähti ympäri ja hävisi taas oviverhon taakse niin nopeasti,

että Aljoša ei ennättänyt sanoa sanaakaan, — vaikka hänen
mielensä olisi kyllä tehnyt sanoa. Hänen mielensä teki pyytää
anteeksi, syyttää itseään, — sanoa edes jotakin, sillä hänen
sydämensä oli täysi eikä hän missään tapauksessa tahtonut lähteä
huoneesta sitä tekemättä. Mutta rouva Hohlakov tarttui hänen
käteensä ja vei itse hänet ulos. Eteisessä hän taas pysähdytti hänet
niinkuin äskenkin.
— Hän on ylpeä, kamppailee itseään vastaan, mutta hän on

hyväsydäminen, ihana, jalomielinen! — lausuili rouva Hohlakov
puoliääneen. — Oi, kuinka minä häntä rakastan, varsinkin toisinaan,
ja kuinka minä nyt taas olen iloinen kaikesta, kaikesta! Rakas
Aleksei Fjodorovitš, te ette ole tietänytkään tätä: tietäkää, että me
kaikki, kaikki — minä, hänen molemmat tätinsä, — kerrassaan
kaikki, myöskin Lise, olemme jo kokonaisen kuukauden ajan vain
sitä toivoneetkin ja pyytäneet, että hän ottaisi eron teidän
suosikistanne Dmitri Fjodorovitšista, joka ei välitä hänestä eikä
rakasta häntä ollenkaan, ja menisi naimisiin Ivan Fjodorovitšin
kanssa, sivistyneen ja oivan nuorukaisen, joka rakastaa häntä
enemmän kuin mitään muuta maailmassa. Meillähän on täällä ihan
oikea salaliitto, ja ehkäpä minäkin vain sen tähden yhä viivyn
täällä…
— Mutta hänhän itki, häntä on taas loukattu! — huudahti Aljoša.
— Älkää uskoko naisen kyyneliä, Aleksei Fjodorovitš, — minä olen

aina naisia vastaan tässä suhteessa, minä olen miesten puolella.
— Äiti, te turmelette ja saatatte perikatoon Aleksei Fjodorovitšin,

— kuului oven takaa Lisen vieno ääni.
— Ei, minä olen syynä tähän kaikkeen, minä olen suuresti

syyllinen! — toisteli lohduton Aljoša piinallisen häpeänpuuskan
vallassa muistellen mielenpurkaustaan ja peittäen kasvonsakin
käsiinsä, niin häntä hävetti.
— Päinvastoin te esiinnyitte kuin enkeli, kuin enkeli, olen valmis

sanomaan sen tuhannen tuhatta kertaa.
— Äiti, minkä tähden hän esiintyi niinkuin enkeli? — kuului taas

Lisen ääni.
— Minusta tuntui jostakin syystä, kun katselin tuota kaikkea, —

jatkoi Aljoša aivan kuin ei olisi kuullutkaan Lisen puhetta, — että hän
rakastaa Ivania, ja niin tulin sanoneeksi tuon tyhmyyden… mitähän
nyt seuraa!
— Kenelle sitten, kenelle? — huudahti Lise. — Äiti, te tahdotte

varmaankin tappaa minut. Minä kysyn teiltä, ettekä te vastaa minulle.
Samassa palvelustyttö juoksi heidän luokseen.
— Katerina Ivanovnan laita on huonosti… Hän itkee… hysteriaa,

on kouristusta.
— Mitä! — huudahti Lise ja hänen äänensä oli nyt levoton.
— Äiti, minä tässä saan hysteriakohtauksen eikä hän!
— Lise, Jumalan tähden, älä huuda, älä tee minusta loppua. Sinä
olet vielä siinä iässä, ettet saa tietää kaikkea mitä aikuiset tietävät,
minä riennän luoksesi ja kerron kaikki mitä sinulle voi kertoa. Voi
hyvä Jumala! Minä riennän, riennän… Hysteerinen kohtaus — se on
hyvä merkki, Aleksei Fjodorovitš, se on mainiota, että hänellä on
hysteerinen kohtaus. Juuri niin pitää ollakin. Minä olen tässä
suhteessa aina naisia vastaan, kaikkia noita hysterioita ja naisten
kyyneliä vastaan. Julia, juokse ja sano, että minä lennän. Mutta että
Ivan Fjodorovitš poistui sillä tavoin, siihen on Katerina Ivanovna itse
syypää. Mutta Ivan Fjodorovitš ei matkusta pois. Lise, älä Jumalan
tähden huuda! Ah, niin, ethän sinä huuda, minähän se huudan, anna
anteeksi äidillesi, minä olen riemastunut, riemastunut, riemastunut!
Mutta huomasitteko, Aleksei Fjodorovitš, millaisena nuorukaisena,
nuorukaisena Ivan Fjodorovitš äsken lähti pois, sanoi kaiken sen ja
lähti! Minä luulin, että hän on tuommoinen tiedemies, akateemikko,
ja yht'äkkiä hän puhui niin kiihkeästi, kiihkeästi, avoimesti ja
nuorukaisellisesti, kokemattomasti ja nuorukaisellisesti, ja kuinka se
kaikki oli kaunista, kaunista, aivan niinkuin te… Ja hän sanoi tuon
saksalaisen säkeenkin, niin, hän on aivan kuin te! Mutta minä
juoksen, juoksen. Aleksei Fjodorovitš, menkää kiireesti toimittamaan
tuo asia ja palatkaa pian. Lise, tarvitsetko jotakin? Jumalan tähden,
älä viivytä hetkeäkään Aleksei Fjodorovitšia, hän palaa kohta
luoksesi.
Rouva Hohlakov juoksi viimein pois. Aljoša aikoi ennen lähtöään

avata
Lisen huoneen oven.
— Ei mitenkään! — huudahti Lise, — nyt ei saa mitenkään tulla!

Puhukaa sieltä oven läpi. Mistä syystä teidät luettiin enkelien
joukkoon? Sen minä vain tahdon tietää.
— Hirveän tyhmyyden tähden, Lise! Hyvästi!
— Ette saa mennä pois! — huudahti Lise.
— Lise, minulla on vakava murhe! Minä tulen kohta takaisin, mutta

minulla on hyvin suuri murhe!
Ja hän juoksi ulos.
6.
Mullerrus tuvassa
Hänellä oli todellakin vakava murhe, senlaatuinen, jommoisia hän

tähän saakka oli harvoin kokenut. Hän oli tuppautunut puhumaan ja
tehnyt »tyhmyyksiä», — ja millaisessa asiassa sitten:
lemmentunteissa! »Mitä minä siitä ymmärrän, miten minä voin
selvitellä tämmöisiä asioita?» — toisti hän itsekseen jo sadannen
kerran ja punastui. — »Ah, eihän häpeä olisi mitään, häpeäminen on
oikea rangaistus minulle, mutta se juuri on onnetonta, että minä nyt
aivan varmasti olen syynä uusiin onnettomuuksiin… Mutta
luostarinvanhin lähetti minut sovittamaan ja yhdistämään. Näinkö
yhdistetään?» Hän muisti taas äkkiä, miten hän oli »yhdistänyt
kädet», ja häntä alkoi taas hirveästi hävettää. »Vaikka minä teinkin
kaiken tämän vilpittömin mielin, niin täytyy vastedes olla viisaampi»,
päätteli hän yht'äkkiä, ja häntä alkoi hymyilyttää oma
johtopäätöksensä.
Katerina Ivanovna oli antanut asian toimitettavaksi Ozernaja-

kadun varrella, ja veli Dmitri asui juuri siellä päin, eräällä sivukadulla
lähellä Ozernaja-katua. Aljoša päätti pistäytyä joka tapauksessa
hänen luonaan ennen menoaan alikapteenin luokse, vaikka
aavistikin, että ei tapaa veljeään. Hän epäili tämän mahdollisesti
tahallaan jostakin syystä nyt piileksivän häntä, — mutta hän päätti,
maksoi mitä maksoi, etsiä hänet käsiinsä. Mutta aika kului: hän ei
ollut minuutiksikaan eikä sekunniksikaan unohtanut kuolevaa
luostarinvanhinta siitä hetkestä asti kuin oli lähtenyt luostarista.
Katerina Ivanovnan antamassa tehtävässä vilahti silmiin eräs

seikka, joka myös kiinnitti tavattomasti hänen mieltään: kun Katerina
Ivanovna mainitsi pienestä koulupojasta, tuon alikapteenin pojasta,
joka oli ääneensä itkien juossut isänsä vieressä, — niin Aljošan
mieleen välähti jo silloin, että tuo poika on varmaankin se äskeinen
koulupoika, joka puri häntä sormeen, kun hän, Aljoša, tiedusti
häneltä, miten oli loukannut häntä. Nyt Aleša oli melkein varma tästä
tietämättä itsekään vielä minkä tähden. Kun näin syrjäseikat vetivät
hänen huomiotaan puoleensa, niin hän tuli hyvälle tuulelle ja päätti
olla »ajattelematta äsken aikaansaamaansa onnettomuutta» ja olla
kiusaamatta itseään katumuksella sekä toimittaa asiansa, kävi miten
kävi. Tätä ajatellen hän tuli taas reippaalle mielelle. Käännyttyään
kadun nurkasta veljensä asuntoa kohti hän tunsi nälkää, otti
taskustaan isältä saamansa vehnäleivän ja söi sen kulkiessaan. Se
vahvisti häntä.
Dmitri ei ollut kotona. Talon isäntäväki — vanha puuseppä, hänen

poikansa ja vanha vaimonsa — katselivat hieman epäluuloisesti
Aljošaa. — Ei ole ollut kolmeen yöhön kotona, kenties on
matkustanut jonnekin, — vastasi ukko Aljošan tarmokkaisiin
tiedusteluihin. Aljoša ymmärsi hänen puhuvan saamiensa ohjeitten
mukaisesti. Hänen kysymykseensä: »Eiköhän hän ole Grušenjkan
luona ja piile taas Tuomaan hoivissa» (Aljoša puhui tahallaan näin
avomielisesti) loi isäntäväki vastaukseksi häneen vain pelästyneen
katseen. »He pitävät hänestä nähtävästi, pitävät hänen puoltaan»,
ajatteli Aljoša, »se on hyvä».
Viimein hän löysi Ozernaja-kadun varrelta pikkuporvarinlesken

Kalmykovin talon, vanhan, kallellaan olevan talorähjän, jossa oli vain
kolme ikkunaa kadulle päin ja likainen piha, jonka keskellä yksinään
seisoi lehmä. Taloon tultiin pihan puolelta eteiseen. Eteisestä
vasempaan asui vanha emäntä tyttärensä kanssa, joka myös oli
vanha eukko. Molemmat tuntuivat olevan kuuroja. Kun Aljoša oli
useampaan kertaan tiedustanut alikapteenin asuntoa, niin toinen
heistä viimein tajusi, että kysyttiin heidän vuokralaisiaan, ja osoitteli
sormellaan eteisen toiselle puolen viitaten oveen, joka vei
siistimpään tupaan. Alikapteenin asunto näytti tosiaankin olevan
aivan yksinkertainen tupa. Aljoša tarttui jo kädellään oven ripaan
avatakseen oven, mutta äkkiä häntä hämmästytti se, että oven
takana oli tavattoman hiljaista. Hän tiesi kuitenkin Katerina
Ivanovnan kertomasta, että virasta erotettu alikapteeni oli
perheellinen mies. »Joko he kaikki nukkuvat tai kenties ovat kuulleet
minun tulevan ja odottavat, kunnes avaan oven. On parasta, että
minä ensin koputan», — ja hän koputti. Kuului vastaus, vaikka ei
aivan heti, vaan ehkä noin kymmenen sekunnin kuluttua.
— Kuka siellä? — huusi joku kovalla äänellä ja hyvin vihaisesti.
Aljoša avasi oven ja astui kynnyksen yli. Hän tuli tupaan, joka oli
jokseenkin tilava, mutta täpö täynnä sekä ihmisiä että kaikenlaista
taloustavaraa. Vasemmalla oli iso venäläinen uuni. Uunin luota
vasemmalla puolella olevan ikkunan luo oli yli koko huoneen
pingoitettu nuora, johon oli ripustettu erilaisia riepuja. Sekä
vasemmalla että oikealla olevan seinän vieressä oli kudotulla
peitteellä peitetty vuode. Toisella niistä, vasemmanpuolisella, oli
päällekkäin neljä karttuunityynyä, toinen toistaan pienempiä.
Oikeanpuolisella vuoteella oli vain yksi hyvin pieni tyyny. Kauempana
etunurkassa oli pieni verholla tai lakanalla erotettu ala, joka oli
muodostettu siten, että poikittain nurkan kohdalle oli pingoitettu
nuora, jonka yli vaate oli heitetty. Tämän verhon takaa näkyi syrjittäin
niinikään vuode, joka oli laitettu lavitsalle ja sen viereen asetetulle
tuolille. Yksinkertainen, puinen, nelikulmainen maalaispöytä oli
siirretty etunurkasta keskimmäisen ikkunan luo. Kaikki kolme
ikkunaa, joissa kussakin oli neljä pientä, vanhuuttaan jo vihertäväksi
käynyttä ruutua, olivat hyvin himmeät ja tarkoin suljetut, niin että
huoneessa oli jokseenkin tukahduttavaa eikä juuri valoisaa. Pöydällä
oli paistinpannu, jossa oli paistettujen munien jätteitä, sekä
nakerrettu leipäpala ja sen lisäksi puolituoppinen, jonka pohjalla oli
vielä hiukan jäljellä ilolientä. Tuolilla vasemmanpuolisen vuoteen
luona oli rouvan näköinen naishenkilö, yllään karttuunipuku. Hänen
kasvonsa olivat hyvin laihat, keltaiset. Hyvin kuopallaan olevat
posket ilmaisivat heti, että hän oli sairas. Mutta kaikkein enimmän
hämmästytti Aljošaa tuon naisraukan katse, joka oli tavattoman
kysyvä ja samalla kertaa hirveän kopea. Ja Aljošan puhuessa
isännän kanssa tämä rouva oli koko ajan vaiti, suuret ruskeat silmät
siirtyen toisesta puhujasta toiseen aina yhtä ylpeän ja kysyvän
näköisinä. Tämän rouvan vieressä vasemmanpuolisen ikkunan
luona seisoi nuori tyttö, jolla oli jokseenkin rumat kasvot ja
punertavat harvat hiukset ja jonka puku oli köyhä, vaikkakin sangen
siisti. Hän tarkasteli ylenkatseellisesti sisäänastunutta Aljošaa.
Oikealla puolella, niinikään vuoteen luona, istui vielä eräs naisolento.
Tämä oli sangen säälittävä ilmiö, noin kahdenkymmenen vuoden
ikäinen nuori tyttö, joka oli kyttyräselkäinen eikä kyennyt liikkumaan,
koska hänen jalkansa, kuten Aljošalle myöhemmin sanottiin, olivat
kuivettuneet. Hänen kainalosauvansa seisoivat hänen vieressään
nurkassa vuoteen ja seinän välissä. Tyttö-raukan ihmeen kauniit ja
hyvyyttä osoittavat silmät katselivat Aljošaa rauhallisesti ja nöyrästi.
Pöydän ääressä istui munan jätteitä lopetellen noin neljänkymmenen
viiden vuoden ikäinen herrasmies, lyhyenläntä, laihanpuoleinen,
heikko ruumiinrakenteeltaan, tukka punertava, parta punainen ja
harva muistuttaen suuressa määrin hajalleen revittyä niinistä
pesuhoskaa (tämä vertaus ja erityisesti sana »pesuriepu» välähti
jostakin syystä heti ensi katsauksella Aljošan mieleen, hän muisti
sen sittemmin). Ilmeisesti juuri tämä herrasmies oli huutanut oven
takaa: »kuka siellä!» sillä muita mieshenkilöitä ei huoneessa ollut.
Mutta kun Aljoša astui sisälle, niin hän ponnahti penkiltä pöydän
äärestä ja pyyhkien nopeasti suutaan reiäkkäällä lautasliinalla kiiruhti
Aljošan luo.
— Munkki kerää varoja luostarille, jopa tiesikin, kenen luo on

tultava! — lausui samassa vasemmassa nurkassa seisova neito
kovalla äänellä. Mutta herrasmies, joka oli juossut Aljošan luo,
pyörähti nopeasti kantapäillään häneen päin ja sanoi kiihtyneellä,
omituisen katkonaisella äänellä hänelle:
— Ei Varvara Nikolajevna, ei se ole sitä, arvasitte väärin. Sallikaa

minun vuorostani kysyä, — lausui hän kääntyen äkkiä taas Aljošan
puoleen, — mikä on antanut teille aiheen tulla… tänne
syrjäsopukkaan?
Aljoša katsoi häneen tarkkaavaisesti, hän näki ensimmäistä kertaa

tämän miehen. Hänessä oli jotakin kulmikasta, hätiköivää ja
ärsyttävää. Hän oli nähtävästi juuri ryypännyt, mutta ei ollut
humalassa. Hänen kasvoissaan kuvastui äärimmäisyyteen menevä
julkeus ja samalla — se oli omituista — ilmeinen pelkuruus. Hän oli
sellaisen ihmisen kaltainen, joka on kauan alistunut ja kärsinyt, mutta
joka äkkiä on hypännyt pystyyn ja tahtoo puolestaan näyttää
olevansa jotakin. Tai vielä paremmin sanoen hän oli niinkuin
ihminen, jonka mieli hirveästi tekisi lyödä teitä, mutta joka hirveästi
pelkää, että te lyötte häntä. Hänen puheessaan ja hänen jokseenkin
läpitunkevan äänensä painossa oli havaittavissa jonkinlaista
hassahtavaa leikillisyyttä, väliin ilkeätä, väliin arkaa, eikä hän
jaksanut pitää yllä aloittamaansa äänensävyä, vaan ääni muuttui
katkonaiseksi. Kysymyksensä »syrjäsopukasta» hän teki ikäänkuin
väristen, silmät pullollaan ja hypähtäen niin lähelle Aljošaa, että tämä
astui koneellisesti askelen taaksepäin. Tämä herrasmies oli puettu
tummaan, sangen huonoon nankinikankaiseen päällystakkiin, jota oli
paikkailtu ja jossa oli tahroja. Hänen housunsa olivat tavattoman
vaaleat, sellaiset, jommoisia ei kukaan ole pitänyt pitkään aikaan,
ruudulliset ja jotakin hyvin ohutta kangasta, alhaalta rypistyneet ja
sen vuoksi ylhäältä kutistuneet, aivan kuin hän olisi pitänyt niitä jo
pikku poikana ja niinkuin sitten ne olisivat jääneet liian pieniksi.
— Minä… olen Aleksei Karamazov… — lausui Aljoša
vastaukseksi kysymykseen.
— Ymmärrän ja tajuan sen varsin hyvin, — lausui heti herrasmies

antaen ymmärtää, että hän muutenkin tiesi, kuka tulija oli. — Minä
taas puolestani olen alikapteeni Snegirev, mutta toivoisin edelleenkin
saavani tietää, mikä nimenomaan on antanut aiheen…
— Minä poikkesin vain omia aikojani tänne. Oikeastaan tahtoisin

sanoa teille sanasen… Jos vain sallitte…
— Siinä tapauksessa on tässä tuolikin, suvaitkaa ottaa paikka.

Vanhanaikaisissa komedioissa sanottiin: »Suvaitkaa ottaa paikka»…
— ja alikapteeni otti nopealla liikkeellä tyhjän tuolin (se oli aivan
yksinkertainen, maalaistyylinen, puinen eikä millään päällystetty)
sekä asetti sen miltei keskelle huonetta. Siepattuaan sitten toisen
samanlaisen tuolin itselleen hän istuutui vastapäätä Aljošaa, nytkin
aivan lähelle häntä, niin että heidän polvensa melkein koskettivat
toisiaan.
— Nikolai Iljitš Snegirev, entinen venäläinen jalkaväen alikapteeni,

vaikkakin paheittensa häpäisemä, niin kuitenkin alikapteeni.
Oikeastaan olisi sanottava: alikapteeni Pahasuinen, sillä vasta
elämäni jälkimmäisellä puoliskolla olen tullut pahaksi suustani. Se
ominaisuus saadaan alennustilassa.
— Niinpä kyllä, — hymähti Aljoša, — mutta saadaanko se

tahtomatta vaiko tahallisesti?
— Jumala tietää, että tahtomatta. En ole koskaan, en koko

elämäni aikana, ollut paha suustani, mutta äkkiä kaaduin ja nousin
pystyyn pahasuisena. Se on korkeamman voiman vaikutusta. Minä
huomaan, että teitä kiinnostavat nykyaikaiset kysymykset. Mutta
millä tavoin minä olen voinut herättää siinä määrin mielenkiintoa,
sillä minähän elän oloissa, joissa ei voi ottaa vieraita vastaan.
— Minä tulin… sen äskeisen asian vuoksi…
— Sen saman asian? — keskeytti alikapteeni kärsimättömästi.
— Tuon teidän kohtauksenne johdosta veljeni Dmitri Fjodorovitšin

kanssa, — lausui Aljoša kömpelösti.
— Minkä kohtauksen. Tuonko saman? Siis pesurievun, niinisen

saunahoskan johdosta? — sanoi alikapteeni työntyen äkkiä niin
lähelle, että heidän polvensa tällä kertaa todella kolahtivat yhteen.
Hänen huulensa puristuivat omituisella tavalla viivaksi.
— Minkä niinihoskan? — mutisi Aljoša.
— Hän on tullut valittamaan sinulle, isä, minua vastaan! —

huudahti verhon takaa nurkasta äskeisen pojan ääni, joka jo oli tuttu
Aljošalle. — Minä purin häneltä äsken sormen! — Verho työntyi
syrjään, ja Aljoša näki äskeisen vihamiehensä nurkassa
jumalankuvien alla, lavitsalle ja tuolille laitetussa vuoteessa. Poika
makasi peitteenään päällystakkinsa ja lisäksi vielä vanha, pumpulilla
täytetty peite. Ilmeisesti hän oli sairas ja palavista silmistä päättäen
kuumeessa. Hän katseli Aljošaa nyt pelkäämättä, toisin kuin äsken:
»Kotona, näes, et pääse kimppuuni.»
— Mitenkä sinä olet sormen purrut? — lausui alikapteeni hypäten

tuoliltaan. — Teiltäkö hän on purrut sormen?
— Niin, minulta. Hän oli äsken kivisillä poikien kanssa kadulla.

Niitä oli kuusi häntä vastaan heittelemässä, ja hän oli yksin. Minä
menin hänen luokseen, mutta hän heitti minuakin kivellä ja sitten
vielä toisen kiven päähäni. Minä kysyin, mitä olen hänelle tehnyt.
Hän hyökkäsi äkkiä kimppuuni ja puri kipeästi sormeani, en tiedä
minkä tähden.
— Minä annan hänelle heti selkään! Tuossa paikassa annan

selkäsaunan, — sanoi alikapteeni, joka oli jo kokonaan hypännyt
tuoliltaan.
— Enhän minä ollenkaan valita, minä vain kerroin… — En

ollenkaan tahdo, että hän saisi selkäänsä. Ja hänhän taitaa nyt olla
sairaskin…
— Luulitteko todellakin, että minä annan hänelle selkään? Ettäkö

minä otan heti Iljušetškan ja pieksän häntä edessänne teidän
suureksi mielihyväksenne? Joko te sitä kohta tahdotte? — lausui
alikapteeni kääntyen äkkiä Aljošaan päin ja tehden sellaisen eleen
kuin aikoisi hyökätä hänen kimppuunsa. — Minun on sääli, hyvä
herra, teidän sormeanne, mutta jos tahdotte, niin mieluummin kuin
pieksän Iljušetškaa minä heti teidän nähtenne oikeutetuksi
tyydytykseksenne hakkaan pois neljä omaa sormeani tällä veitsellä.
Luullakseni neljä sormea riittää teille kostonhimonne tyydyttämiseksi
ettekä vaadi enää viidettä?… — Hän pysähtyi äkkiä aivan kuin olisi
tukehtumaisillaan. Jokainen piirre hänen kasvoissaan liikkui ja
nytkähteli, ja hän oli hyvin uhmailevan näköinen. Hän oli kuin
haltioissaan.
— Nyt luulen ymmärtäneeni kaiken, — vastasi Aljoša hiljaa ja

surullisesti istuen edelleen paikallaan. — Teidän poikanne on siis
hyvä poika, hän rakastaa isäänsä ja hyökkäsi minun kimppuuni,
koska olen sen miehen veli, joka on teitä loukannut… Minä
ymmärrän nyt sen, — toisti hän miettivänä. — Mutta veljeni Dmitri

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Junqueiraâ€™s Basic Histology Text

and Atlas - eBook PDF

Anthony L. Mescher, PhD

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1 Histology & Its Methods 5 Connective Tissueâ•‡ 96

2 The Cytoplasmâ•‡ 17 7 Cartilageâ•‡129

Neural Plasticity & Regenerationâ•‡ 187 15 Digestive Tractâ•‡ 295

17 The Respiratory Systemâ•‡ 349

Summary of Key Pointsâ•‡ 411 22 The Female Reproductive Systemâ•‡ 460

H istology is the study of the tissues of the body and

FIGURE 1–1â•‡ Sectioning fixed and embedded tissue.

(a) Fixation Dehydration Clearing Infiltration Embedding

Embedding & Sectioning Staining

FIGURE 1–3â•‡ Components and light path of a Fluorescence Microscopy

Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

FIGURE 1–4â•‡ Appearance of cells with fluorescent microscopy.

FIGURE 1–5â•‡ Unstained cells’ appearance in three types of light microscopy.

the specimen allows them to be digitally reconstructed into a

Histology & Its Methods of Study â•‡ ■â•‡ Light Microscopy

Although a very small spot of light originating from one plane

power of the objective lens. Confocal microscopy (Figure 1–6)

FIGURE 1–8â•‡ Electron microscopes.

Electron gun Electron gun

TEM image Lens SEM image

(a) Transmission electron microscope (b) Scanning electron microscope

Histology & Its Methods of Study â•‡ ■â•‡Autoradiography

FIGURE 1–9â•‡ Microscopic autoradiography.

and preparing to divide. Dynamic events may also be analyzed.

Examples of molecules that interact specifically with

FIGURE 1–11â•‡ Immunocytochemistry techniques.

FIGURE 1–12â•‡ Cells and tissues stained by immunohistochemistry.

throughout the cytoplasm. Primary antibodies against the

Specific cytokeratins Tumors of epithelial origin

FIGURE 1–14â•‡ Interpretation of 3D structures in 2D sections.

Histology & Its Methods of Studyâ•‡ SUMMARY OF KEY POINTS

Preparation of Tissues for Study Autoradiography

Histology & Its Methods of Studyâ•‡ ASSESS YOUR KNOWLEDGE

C ells and extracellular material together comprise all

abundant in the outer half, while phosphatidylserine and

FIGURE 2–1â•‡ Lipids in membrane structure.

The Cytoplasmâ•‡ ■â•‡ The Plasma Membrane

General structure of a phospholipid Cholesterol

Sugar chains of a glycolipid

FIGURE 2–2â•‡ Proteins associated with the membrane lipid bilayer.

Sugar chain Sugar chain

FIGURE 2–3â•‡ Membrane proteins.

The Cytoplasmâ•‡ ■â•‡ The Plasma Membrane

Functions of Plasma Membrane

hormones, are integral membrane proteins at the cell

FIGURE 2–5â•‡ Major mechanisms by which molecules cross membranes.

FIGURE 2–6â•‡ Three major forms of endocytosis.

The Cytoplasmâ•‡ ■â•‡ The Plasma Membrane

and then internalize this material into a cytoplasmic vacuole or

■âŒ€ In autocrine signaling, signals bind receptors on the

FIGURE 2–7â•‡ Receptor-mediated endocytosis involves regulated membrane trafficking.

FIGURE 2–8â•‡ Major types of membrane receptors.

The Cytoplasmâ•‡ ■â•‡ Cytoplasmic Organelles

Inactive protein Active protein

a Channel-linked receptors b Enzymatic receptors

2 G protein binds to activated Inactive protein

›â•ºCYTOPLASMIC ORGANELLES energy. Oxygen, CO2, electrolytic ions, low-molecular-weight

FIGURE 2–9â•‡ Polyribosomes: free or bound to the endoplasmic reticulum.

Specific proteins imported to Secretory vesicles Lysosomes