List the different microscopes, function/use, o includes a computer-driven mirror system (the

and distinct feature. beam splitter) to move the point of illumination

across the specimen automatically and rapidly
MICROSCOPES
Polarizing Microscope
- usually used in the field of geology or
LIGHT MICROSCOPES
petrography but may also be used in medicine,
-based on the interaction of light with tissue
chemisty, biology, and metallurgy
components
- commonly used specimens are opaque
-used to reveal and study tissue features
materials like rocks, minerals, wood, urea etc.
BRIGHT-FIELD MICROSCOPE
- has a polarizer and an analyzer which enables
-stained tissue is examined with ordinary light
the microscope to employ reflected and
passing through the preparation
transmitted light
-optical components are the condenser focusing
- helpful in obtaining information regarding the
light on the object to be studied; the objective
color absorption, structure, composition, light
lens enlarging and projecting the image of the
refraction of various specimens.
object toward the observer; and the eyepiece (or
ocular lens) further magnifying this image and
ELECTRON MICROSCOPE
projecting it onto the viewer’s retina or a charge-
- are microscopes that use beams of electrons
coupled device (ccd) highly sensitive to low light
to illuminate the specimens and acquire a more
levels with a camera and monitor
detailed image.
FLUORESCENCE MICROSCOPE
- has two types: Transmission Electron
o when certain cellular substances are irradiated
Microscope and Scanning Electron Microscope
by light of a proper wavelength, they emit light
with a longer wavelength—a phenomenon called
Transmission Electron Microscope
fluorescence
- used to view thin specimens and acquire an
o tissue sections are usually irradiated with
image of its interior components
ultraviolet (uv) light and the emission is in the
- uses transmitted electrons to create an image
visible portion of the spectrum
PHASE-CONTRAST MICROSCOPES
Scanning Electron Microscope
o uses a lens system that produces visible
- used to view a more detailed view of the
images from transparent objects and,
surfaces of the specimens and other
importantly, can be used with living, cultured
components that are not possible through TEM.
cells
- uses a focused electron beam to scan the
o based on the principle that light changes its
surface of the specimen in a raster pattern
speed when passing through cellular 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 other
CONFOCAL MICROSCOPES
o stray (excess) light reduces contrast within the
image and compromises the resolving power of
the objective lens, confocal microscopy 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
List and define 15 terms in microscopy.
Aperture
-a measure of the resolving power of the power
of an objective respectively.
Achromatic lens
-A lens which brings in light from two parts of the
spectrum (red and blue wavelengths) to the
same focus, reducing Chromatic Aberration.
This is the most common lens on a microscope.
Magnification
- the enlargement of an object through the lens
system. This is determined by multiplying the
magnifying power of the objective by the
eyepiece.
Parfocal (parfocality)
- a term used to describe the property of a
microscope where the subject stays in focus
when the objective lenses are changed.

Coaxial
- focusing system where the coarse and fine
focus are mounted together on a common axis.
Condenser (substage)
- provides an even cone of light that illuminates
the specimen. Light from the condenser
converges on the specimen, passes through it,
and diverges to from an inverted illumination
cone that is captured by the objective lens
Chromatic Aberration
- failure of lens to bring light of different
wavelengths to a common focus.
Depth of Field
- the distance along the optical axis throughout
which the object can be located and yet be
imaged with satisfactory clarity.
Resolution
- a measure of the ability of a lens to image
closely spaced objects so they are recognized
as separate objects.
Resolving power
- the capacity of any optical system to
distinguish and separate details in a specimen.
Working Distance
- the distance between the cover glass or object
and the tip of the objective. This governs the
allowable movement of the objective in obtaining
critical focus of the specimen.

Eyepiece
- also known as ocular, it produces the second
stage of magnification enlarging the image
magnified by the objective lens.

Field-Of-View
- the visible area through the eyepiece when the
microscope is in focus.
Immersion Objective
- most common is the 100x oil objective. Oil is
placed on the cover glass of the slide which (and
sometimes on the top element of the condenser)
to produce a high magnification and high
resolving power of the objective when immersed
in the oil. This produces the full NA of objective
lens.
List the different visualization techniques
and explain.
IMMUNOHISTOCHEMISTRY
- sample visualization done after incubation with an
antibody
- principle:
o Immune cells of the body produces ANTIBODIES
which interact and reacts with ANTIGENS of “foreign”
molecules.
- basic process:
o Tissue section which is believed to have the protein
of interest is secured
o Tissues section is incubated in solution with
antibody
o The antibody, stained by fluorescent dye, will bind
to the protein and will be able to be viewed under a
light electron microscope
- two detection techniques:
o DIRECT IMMUNOHISTOCHEMISTRY
▪ generally one step and involve the use of directly
labelled antibodies to detect antigens of interest within
the tissue
▪ straightforward and less time-consuming but are
usually less sensitive and lack the ability to amplify
weak signals
▪ primary antibody is already labelled
• range of labelled primary antibodies is comparatively
limited
o INDIRECT IMMUNOHISTOCHEMISTRY
▪ utilizes an unlabelled primary antibody to detect the
antigen of interest in the tissue
▪ then, a secondary labelled antibody is used to bind
to the primary antibody
▪ amplifies relatively weak antigen signals in tissue as
many secondary antibodies can bind to different
antigenic sites of the primary antibody
• this is why indirect IHC is preferred over direct IHC
HYBRIDIZATION
- technique for precise detection and localization of a
specific nucleic acid sequence within a histologic
section
- purpose:
o determines if cell has a specific sequence of DNA
o identifies the cells with the specific RNA
o determines the localization of gene in a specific
chromosome
- uses complementary probes to specifically localize
DNA and RNA sequences within a section of tissue
(in situ) or if small enough, an entire tissue, like an
embryo
Cell fractionation - process used to separate cellular
components while preserving individual functions of
each component
- With this, scientists were able to know the functions
of organelles
- Centrifugation is necessary to separate components
Centrifugation - separates components based on
size and density
- The smaller components stay homogenized in the
liquid
- Larger components will move to the bottom
- Repeating the centrifugation with increasing force
allows smaller cellular components to be separated
- denser and larger components are the first ones to
settle at the bottom at each repeat of centrifugation
Microscopic autoradiography
- method of localizing newly synthesized
macromolecules in cells or tissue sections.
- Radioactively labeled metabolites (nucleotides,
amino acids, sugars) provided to the living cells are
incorporated into specific macromolecules (DNA,
RNA, proteins) and emit weak radiation that is
restricted to those regions where the molecules are
located
- Application: tracking the pathway of proteins from
site of synthesis to deployment to target organs
Cell culture
- widely used to study molecular changes that occur
in cells
- Keeping cells alive to for observation
- Application: vaccines
Enzyme histochemistry
- method of localizing cellular structures using a
specific enzymatic activity present in those structures
- Examples of enzymes that can be detected through
histochemistry: phosphatases, dehydrogenases,
peroxidase
Steps in tissue processing. Explain how
each step is done.
- Tissue processing is a procedure that is done
between tissue fixation and the embedding or
sectioning of paraffin blocks.
The 2 main steps in tissue processing:
1. Sectioning of tissue = most tissues and
organs are too thick for light to pass through,
thin translucent sections are cut from them and
placed on glass slides for microscopic
examination of the internal structures
- FROZEN SECTIONING:
- It is a rapid way of fixing and mounting a
histology section using a microtome calles a
"cryostat" in a cabinet at subfreezing
temperature.
- It is used to section the block with tissue,
and the frozen sections are placed on slides for
rapid staining and microscopic examinations.
- PARAFFIN SECTIONING
- Microscopic slides are produced from
tissues that were taken from the body for
diagnosis of disease processes which are then
processed in the histopathology laboratory.
Basic steps:
a. Fixation
- Small pieces of tissue are placed in solutions
of chemicals known as "fixatives" that cross-link
proteins and inactive degradative enzymes that
preserves cell and tissue structure.
- These fixatives must fully diffuse through the
tissues to preserve all cells and are often
introduced via blood vessels.
b. Dehydration
- The tissue is transferred through a series of
increasingly concentrated alcohol in 100% which
removes all the water inside.
c. Clearing
- Alcohol is removed in organic solvents in which
both alcohol and paraffin are miscible.
- The most common clearing agent is toluene.
d. Infiltration
- The tissue is placed in melted paraffin until it
becomes completely infiltrated with this
substance and replaces clearing agents such as
toluene.
e. Embedding
- The paraffin-infiltrated tissue is placed in a
small mold with melted paraffin and is allowed to
harden.
f. Trimming
- The resulting paraffin block is trimmed to
expose the tissue for sectioning (slicing) on a
microtome.
---------------------------------------------
2. Staining of tissue = most cells and
extracellular material are completely colorless;
thus, this method make various tissue
components distinguishable from one another
- BASIC DYES:
- oluidine blue, alcian blue, and methylene
blue
- the main tissue cpmponents that ionie and
react with basic dyes do so because of acids in
their composition (DNA, RNA and
glycosaminoglycans)
- ACID DYES
- eosin, orange G, and acid fuchsin
- stains the acidophilic components of
tissues such as mitochondria, secretory
granules and collagen
- The combination of the stains ematoxylin
and eosin (H&E) is the most commonly used
staining method that acts as basic and acidic
dyes, respectively.