Chapter 1 - Histology and its methods of study Histology: study of tissue -involves all aspects of tissue of biology Focus:

how cells structure and arrangement optimize functions specific to each organ Two interacting components: cells and intercellular matrix Extracellular matrix: consists of many kinds of molecules, highly organized and forms of complex structures, such as collagen fibrils, and basement membranes Function of e.m.: furnish mechanical support for the cell, carry away catabolites and secretory products. Cells produce e.m. while e.m. influence / control the cells by its molecules Therefore there are intense interactions between them Preparation of tissues for study 1. Fixation - Autolysis: tissue digestion by enzymes present within the cells - Can be done chemically and physically - Chemically using fixatives - Formalin: one of the best fixatives for routine light microscopy (buffered isotonic solution of 37% formaldehyde) 2. Embedding (tissue impregnation) - gives a rigid consistency to the tissue - uses paraffin (light microscope) and plastic

resins (light and electron microscope) - plastic embedding : prevents shrinking effect & gives lesser or no distortion to the cells - two main steps : a. dehydration o Mixtures of ethanol and water (70% to 100% ethanol) b. Clearing 3. Sectioning - sectioning uses microtome (steel or glass) - uses nm or angstrom as a unit of measurement (1nm = 1/1,000 micrometer = 10-6 mm = 10-9 m // 1 angstrom = 0.1 nm or 10-4 micrometer) - rapid freezing sectioning : physical not chemical uses a cryostat (freezing microtome) - it is routinely used in hospitals to study specimens during surgical procedures - its effective for sensitive enzymes since it will not activate most of the enzymes - using physical method is useful in studying lipids since it will not dissolve the lipids in the fixed tissues 4. Staining - used not only to distinguish but also to differentiate or to make distinctions between the tissue molecules - uses dyes (physical) and other chemical solution (chemical) to stain tissue

basophilic for net negative charge and cationic components such as proteins with many ionized groups for acidophilic - dyes for basophilic : toluidine blue, alcian blue, and methylene blue (nucleic acids, glycosaminoglycans, and acid glycoproteins) - dyes for acidophilic : orange G, eosin, acid fuchsins (mitochondria, sercretory granules and collagen) - hematoxylin and eosin = most common, thrichomes = histology, picrosirius = collagen - counterstain used to give more information (enhances the stain by allowing a better recognition of nuclei or other structures) - lipid soluble dyes : avoid the removal of lipids (Sudan black) - metal impregnation technique for nervous tissues A. Microscopy I. Light microscopy a. Bright-field - widely used for students of histology - condenser : collects & focuses light // objective lenses : enlarge and project the illuminated image of the object in the direction of the eyepiece // eyepiece (ocular lens) : further magnifies the image and projects it into the viewers retina

magnifying power = objective lens x ocular lens resolving power smallest distance that between two particles at which they can be seen as separated the quality of the image (clarity and richness of details) depend on its resolving power magnification does not improve resolution it only enlarges the resolved view of a specimen by its objective

b. Confocal - stray light in a regular bright-field microscope reduces the contrast within the image and compromises the resolving power of the objective lens but in confocal microscopy, it avoids the stray light which resulted to a great improvement in its resolution and allowed the localization of specimens components with much greater precision c. Fluorescence - Fluorescence: substances irradiated by light of a proper wavelength, they emit light with a longer wavelength - Uses UV light - Fluorescent substance appear brilliant on a dark background d. Polarizing

allows the recognition of structures made of highly organized molecules - appear as bright structure against a dark background - birefringence : ability to rotate the direction of vibration of a polarized light e. Phase-contrast - uses lens system that produces visible images from transparent objects changes the speed of light when it passes through structures with different refractive indices - does not require fixation or staining - effective for living cells or tissue cultures f. Differential interference - produces threedimensional aspect of a phase-contrast microscopy II. Electron microscopy a. Transmission electron b. Scanning electron III. Autoradiography B. Cell and tissue culture C. Histochemistry & Cytochemistry