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Cell bio 1
Scopes and stains
44
Biology
Undergraduate 4
02/26/2008

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Cards

Term
what are the 5 types of light microscopes?
Definition

bright field

dark field

phase contrast

fluorescence

confocal

Term
how does a bright field scope work?
Definition
light passes through object into lens
Term
what is a compound scope
Definition
has two lenses
Term

how does a dark field scope work

Definition

scattered light (ex. from side) is viewed

Term

what type of scope is used to view live cells

Definition
phase contrast
Term
what are the two main components of a phase contrast scope
Definition

phase ring

phase condensor

Term

what are the 3 main parts of a fluorescence scope

Definition

excitation filter

dichroic mirror

barrier filter

Term
what do fluoresenct molecules do
Definition
excite light of a specific wavelength to another specific wavelength
Term
what type of scope combines multiple planes of focus
Definition
confocal
Term
what scopes use fluorescent stains
Definition

fluorescence and

confocal

Term
what are the two main components of a confocal scope
Definition

pinhole opening

computer

Term
how are images from an electron scope generated
Definition
by the scattering ofan electron beam
Term

what is a microtome? what does it do

Definition

instrument for preparing electron scope samples

1. replaces water in tissues w/parafin wax

2. slices samples at 1-10 micrometers

Term

why is wax used in some sample preps for electron scopes

Definition
to replace water in samples, hardening them enough to be sliced
Term
what type of electron microscope sample prep would be used for a high water sample
Definition
cryosectioning
Term
why must cryosectioning freezing be done quickly 
Definition
so ice crystals don't form that would expand and break tissues
Term
what is the diff between TEM and SEM
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

Term
how are most SEM samples prepped? when can't this be done
Definition

coated in heavy metals

-can't be done when sample was cryosectioned

Term

what is freeze-fracturing?

what is used most often to view?

Definition

when tissue samples are frozen rapidly then cracked, giving cross section.

-used often to view cell membrane structures

Term
what are the three stages associated with staining
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

Term
what are used to permeabilize cells. examples
Definition

detergants

NP-40

Triton-x

Term
what are used to fix cells
Definition

aldehydes and

alcohols

Term
what is a vital stain? examples
Definition

stain that does not kill cells right away

Green Fluorescent Protein

Nile Blue

Term
how is GFP used to stain a protein
Definition

protein of interest and GFP inserted in plasmid

plasmid introduced to living tissue

fusion protein expressed. 'tagged'

Term
what is a fusion protein
Definition
GFP tagged to a protein of interest
Term
what is a mordant
Definition
an additive needed with some dyes to make them dry quickly or set
Term
what are examples of organic dyes? what do they stain
Definition

fuchsin - mitochondria

sudan black - fat granules

coomassie blue - protein

hematoxylin/eosin - nucleus/cytoplasm

Term

what are the two types of fluorescent stains

Definition

general fluorescent stains

specific markers

Term
what are 2 examples of general fluorescent stains? what do they stain?
Definition

DAPI - stains DNA (nuclear stain)

Acridine Orange - nucleic acid stain

(DNA green RNA orange)

Term
what stain would you use to stain RNA
Definition
acridine orange
Term
how do specific marker fluorescent stains work?
Definition
fluorescent molecules attached to antibodies specific to certain structures/molecules
Term
what type of stain is fluorescein
Definition
specific marker fluorescent stain
Term
where use of column purification was discussed
Definition
to purify antibodies
Term

what type of light needed to view:

fluorescein

rhodamine

DAPI

ethidum bromide

Definition

fluorescein - blue light

rhodamine - green/yellow

DAPI - UV

ethidium bromide - UV

Term
what type of immunofluorescence staining uses a single antibody with a fluorescent tag
Definition
direct
Term
what type of immunofluorescence staining uses a primary and secondary antibody
Definition
indirect
Term
why does using indirect immunofluorescence produce a stronger signal
Definition
multiple secondary Abs can attach to the primary
Term
what is negative staining? what type of microscopy most commonly used for?
Definition

staining with heavy metals either directly or with Abs

-used largely with TEM

Term
what are the advantages to negative staining?
Definition

can use whole specimens

don't need to section

Term
what are the two main staining techniques for electron microscopy? 
Definition

negative staining

immunogold

Term

what is immunogold labeling

Definition

technique for labeling electron microscope samples.  primary Ab for specific sample, secondary Ab for primary

Term
what is the disadvantage to immunogold labeling
Definition
only molecules on surface of section can be seen, Abs can't penetrate resin used to hold sections
Term
what type of electron microscope staining needs sectioned samples
Definition
immunogold labeling
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