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34 Cards in this Set
- Front
- Back
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Light Microscopy
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0.2 microns
Practical: 1 micron 1500X Magnification |
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Types of Light Microscopy
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Phase contrast
Enzyme Cytochemistry Fluoresence immunoflurorescence Confocal laser scanning |
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SEM
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2.5 nm
Practical: 10nm Renders depth of field |
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TEM
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1 nm
100,000X Magnification |
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Resolution of Atomic Force Microsope
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0.05 nm
Cells may be living |
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Resolution
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Smallest distance between two particles at which the particles can be discerned as separate objects
Directly proportional to wavelength (higher wavelength, better resolution) |
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Size Markers
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Ovum - 100 microns
RBC - 7-8 microns Bacteria - 200 nm Ribosome - 15 nm Membranes- 7-10 nm |
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Preparing a Sample
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Fixation
Embedding Sectioning Staining Observation |
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Fixation
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crosslink proteins with formalin/formaldehyde - cross-links proteins, especially lysine residues- or gluteraldehyde - denatures protein
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Embedding
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Samples are dehydrated in a graded alcohol series follwed by clearing of dehydrant with toluene or xylene. Put in paraffin or plastic molds.
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Sectioning
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Cut with a sharp knife and put on either a slide for LM, a copper grid for TEM, or on studes for SEM
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Ground sections
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Calcified tissue must be decalcified, sectioned, stained, or physically ground down
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Histochemical and cytochemical stains
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target specific molecules, receptors, or enzymes
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Immunocytochemical
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based on antibody specificity
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TEM-bound samples
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stained with OsO4, lead citrate, or uranyl acetate - increases contrast
Conjugate gold or ferritin to antibodies or ligands to be used to localize specific molecules or receptors |
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Autoradiography
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uses radioactively labelled probes
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Freeze-fracture
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Prepare intramembrane surfaces for TEM
P-face (interior protoplasmic) retains integral proteins E-face (extracellular monolayer) has pits from where proteins were pulled |
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Hematoxylin
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basophilic
positively charged dye blue Stains nucleic acids |
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Eosin
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acidophilic
negatively charged dye pink stains proteins |
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Modified Romanovsky (Wright; May-Grunwald-Giemsa)
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basic - methylene blue
metachormatic basic - azure acidic - eosin For blood and bone marrow: dark nuclei, pale cytoplasm, granule-specific color |
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Periodic acid-schiff
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Stains carbs bright red/magenta
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Mallory trichrome
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collagen/reticular fibers- bright blue
nuclei - bright red mucinogens - clear muscle & RBCs - red/orange |
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Masson's trichrome
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collagen - green/light blue
cytoplasmic proteins - red basophilic - dark blue |
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Toluidine blue
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nucleic and cytoplasmic acids - blue
acidic mucopolysaccharides (mast cells, cartilage) -purple in thin plastic sections |
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van Gieson
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Trichome
collagen - red cytoplasmic protein - yellow elastin - black |
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OsO4
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black/brown in LM - reduced by unsaturated fatty acids
e- dense in TEM (myelin) |
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Fe-hematoxylin
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Cytological detail
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Phosphotungstic acid - hematoxylin (PTAH)
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fibers
intracellular muscle & glial - dark blue Extraceulluar collagen and elastic - red/brown nuclei and RBCs - blue |
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Alcian blue
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mucopolysaccharides (secretory mucins) - bright blue
fast red - nuclei are red |
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Resorcinol-fuchsin
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elucidates elastic fibers
use fast red for nuclear counterstain |
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Feulgen
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histochemical reaction for DNA
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Weigert
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elastic fibers - black/blue
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Nissl
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Basic dyes stain ribosomes and nuclei
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Carmine
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red for glycogen or nuclei
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