Term
| what are the 5 types of light microscopes? |
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Definition
bright field dark field phase contrast fluorescence confocal |
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Term
| how does a bright field scope work? |
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Definition
| light passes through object into lens |
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Term
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Definition
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Term
how does a dark field scope work |
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Definition
scattered light (ex. from side) is viewed |
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Term
what type of scope is used to view live cells |
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Definition
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Term
| what are the two main components of a phase contrast scope |
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Definition
phase ring phase condensor |
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Term
what are the 3 main parts of a fluorescence scope |
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Definition
excitation filter dichroic mirror barrier filter |
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Term
| what do fluoresenct molecules do |
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Definition
| excite light of a specific wavelength to another specific wavelength |
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Term
| what type of scope combines multiple planes of focus |
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Definition
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Term
| what scopes use fluorescent stains |
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Definition
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Term
| what are the two main components of a confocal scope |
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Definition
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Term
| how are images from an electron scope generated |
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Definition
| by the scattering ofan electron beam |
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Term
what is a microtome? what does it do |
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Definition
instrument for preparing electron scope samples 1. replaces water in tissues w/parafin wax 2. slices samples at 1-10 micrometers |
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Term
why is wax used in some sample preps for electron scopes |
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Definition
| to replace water in samples, hardening them enough to be sliced |
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Term
| what type of electron microscope sample prep would be used for a high water sample |
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Definition
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Term
| why must cryosectioning freezing be done quickly |
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Definition
| so ice crystals don't form that would expand and break tissues |
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Term
| what is the diff between TEM and SEM |
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Definition
TEM - image formed from electrons that pass through sample. 2-D image SEM - image formed from electrons that deflect off of sample. 3-D image |
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Term
| how are most SEM samples prepped? when can't this be done |
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Definition
coated in heavy metals -can't be done when sample was cryosectioned |
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Term
what is freeze-fracturing? what is used most often to view? |
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Definition
when tissue samples are frozen rapidly then cracked, giving cross section. -used often to view cell membrane structures |
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Term
| what are the three stages associated with staining |
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Definition
1. sample prepped (w/microtome or cryosectioning) 2. permeabilized - detergants used to put holes in cell membrane 3. fixed - freeze or hold cells at particular stage. freeze cellular components |
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Term
| what are used to permeabilize cells. examples |
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Definition
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Term
| what are used to fix cells |
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Definition
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Term
| what is a vital stain? examples |
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Definition
stain that does not kill cells right away Green Fluorescent Protein Nile Blue |
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Term
| how is GFP used to stain a protein |
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Definition
protein of interest and GFP inserted in plasmid plasmid introduced to living tissue fusion protein expressed. 'tagged' |
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Term
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Definition
| GFP tagged to a protein of interest |
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Term
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Definition
| an additive needed with some dyes to make them dry quickly or set |
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Term
| what are examples of organic dyes? what do they stain |
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Definition
fuchsin - mitochondria sudan black - fat granules coomassie blue - protein hematoxylin/eosin - nucleus/cytoplasm |
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Term
what are the two types of fluorescent stains |
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Definition
general fluorescent stains specific markers |
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Term
| what are 2 examples of general fluorescent stains? what do they stain? |
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Definition
DAPI - stains DNA (nuclear stain) Acridine Orange - nucleic acid stain (DNA green RNA orange) |
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Term
| what stain would you use to stain RNA |
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Definition
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Term
| how do specific marker fluorescent stains work? |
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Definition
| fluorescent molecules attached to antibodies specific to certain structures/molecules |
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Term
| what type of stain is fluorescein |
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Definition
| specific marker fluorescent stain |
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Term
| where use of column purification was discussed |
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Definition
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Term
what type of light needed to view: fluorescein rhodamine DAPI ethidum bromide |
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Definition
fluorescein - blue light rhodamine - green/yellow DAPI - UV ethidium bromide - UV |
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Term
| what type of immunofluorescence staining uses a single antibody with a fluorescent tag |
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Definition
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Term
| what type of immunofluorescence staining uses a primary and secondary antibody |
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Definition
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Term
| why does using indirect immunofluorescence produce a stronger signal |
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Definition
| multiple secondary Abs can attach to the primary |
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Term
| what is negative staining? what type of microscopy most commonly used for? |
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Definition
staining with heavy metals either directly or with Abs -used largely with TEM |
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Term
| what are the advantages to negative staining? |
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Definition
can use whole specimens don't need to section |
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Term
| what are the two main staining techniques for electron microscopy? |
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Definition
negative staining immunogold |
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Term
what is immunogold labeling |
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Definition
technique for labeling electron microscope samples. primary Ab for specific sample, secondary Ab for primary |
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Term
| what is the disadvantage to immunogold labeling |
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Definition
| only molecules on surface of section can be seen, Abs can't penetrate resin used to hold sections |
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Term
| what type of electron microscope staining needs sectioned samples |
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Definition
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