Term
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Definition
| d=(.61λ/nsinα) d is the resolution limit, n is refractive index α is the angle at the apeture denominator is the NA or numerical apeture |
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Term
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Definition
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Term
| ways to enhance contrast without staining hint LM |
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Definition
| phase contrast, differential, interference contrast DIC, Darkfield microscopy |
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Term
| preparation of tissue for microscopy: Step in order |
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Definition
| Fixation, Dehydration, Infiltration and embedding, Sectioning, Staining |
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Term
| Resolutation limits for: Eye, LM, EM |
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Definition
| H: 0.2mm, LM: 0.2um, EM ~2nm |
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Term
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Definition
| prevent postem changes (ie, autolysis, protein denature, degradation); prepares for subsequent proceedures, increases refractive index, increases affinity for stains |
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Term
| 2 broad classes of fixatives (which is more frequently used) |
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Definition
| Cross-linking aldehydes (more common) precipating (alcholols acetic acid |
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Term
| 2 types of aldehydes and what are their pros and cons |
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Definition
| Formaldehyde: binds amino groups 2 steps weakly used for LM; Glutaraldehyde: used in combo with formaldehyde strongle crosslinks amino groups in a single step, used frequently for EM (Both forms will result in the washing out of lipids) |
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Term
| What fixative is used to preserce cell lipids |
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Definition
| osmium tetroxide (OsO4) stains brown electron dense used for LM and EM |
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Term
| Dehydration what are the steps what does is do |
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Definition
| embedding material not permissible in water gradual immersion prevents shrinking and extraction artifaction, final graded emersion in a more compatible embedding medium is required ie toluene or xylene these are called transition solution |
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Term
| Infiltation and Embedding, 2 mediums frequently used and their pros and cons |
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Definition
| allows for sections must be infultrated with medium that is solid at room temp for several hours; Parafin: cuases more spatial distortion but permits better staining, plastic procides better reolution of tissue being that it can be cut thinner less hydrophilic most plastics dont stain well with aques based stains |
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Term
| Sectioning thickness for LM and EM, microtone blade uses |
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Definition
| 8-10um with parafin on a metal knife, 1 um thick with plastic for LM with glass or diamon chuck, plastic cuts down to 60-80nm with correct blade EM |
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Term
| Stains for LM most commonly used |
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Definition
| H and E, hematoxylin: BLUE base like (dye +) stains anions like DNA RER hemachromatin nucleoli and phosphates groups, Eosin: acid like Dye - PINK stains cations substances with amino groups such as proteins |
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Term
| what is an artifact and what leads to them |
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Definition
| alteration in the tissue that result from processing IE shrinkage folds and tears, during transition (clearing) through xylene or toluene and inflitation in parafin all lipids are extracted unless fixed with OsO4 which would classify as an artifact |
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Term
| "stains for EM" and there traits how do they function dark/ light regions? |
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Definition
| uses a beam of electrons that pass through a sample scatering from metal salts gives the appearance of dark sections e- dense, spots with not e- scattering apear light or "lucent" ex: uranyl acetate-binds poitive charges (phosphoproteins) lead citrate (binds positive charge structions, OsO4 binds lipids |
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Term
| LM vs EM advantages in image stains resolutation magnification sample size |
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Definition
| LM polychromatic, multeple stains, low res, 1000x,large, EM monochromatic, few stains, high res, 200,000x, smal sample size |
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Term
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Definition
| used with TEM, sam[le is quick przen and mechanically fractured coated with metal (replica) is used for EM used to study membrane proteins |
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Term
| Cytochemistry stains, their mode of action & uses |
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Definition
| Feulgen reaction: stains DNA, HCL splits off purine bases and exposes aldehyde grops on the Deoxyribose sugars which and then react with schiffs reagent to form fuchia colored reaction, PAS (period acid Schiff), stains carbohydrates, periodic acid oxidizes adjeacent hydroxyl groups ( on hexose carbs to aldehydes which can then be reacted with Schiffs reagent also fuchia Know your target!! same color as feulgen |
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Term
| Immunohistochemistry examples, function pros and cons |
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Definition
| specific maolecules with tissue may be visuallized by incuabting the tissue with antibodies that have been generated against the molecule, this is the primary target, the primary antibodies are a target for the secondary antibodies that have a reporter attatched (enzyme linked, fluoresent chromophore, or electron dense gold) ezymative can be used for LM limited number of reporters and background endogenous enzyme activity may be a problem, flourescent allows multeple simultainous targets, colloid gold specifically used for EM is enhanced with silver salts |
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Term
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Definition
| targets specific mRNA or DNA strands with some form of reporter |
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