Exam1Lec1Microscopy Flashcards
Wht are the steps of preparing a sample to be looked at under a microscope?
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- Fixation
- Dehydration
- Clearing
- embedding
- sectioning
- mounting
- staining
Fixation
preservation so it stabalizes the protein to prevent degradation
What fixation chemical method is used for LM?
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Formaldehyde solution to react with amine group
What fixation chemical method is used for EM?
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Glutaraldehyde solution that cross links proteins and uses osmium tetroxide that preserves membranes
Dehydration
get water out by bathing tissue in a progressive series of ethanol + water mixture up to 100% ethanol so you can embedd it
Clearing
100% ethanol with the tissue is replaced by xylene and makes the tissue transparent
this allows paraffin to penetrate the tissue, xylene is miscible w/ the embedding medium which makes the tissue clear
embedding
embeds the tisue in a hardend mold for easy slicing in 2 types waxy substance. Paraffin and epoxy resins (used when tissue is cleared with plastic solvents)
What are the steps of using paraffin wax to embedd?
- Paraffin is melted to 58-60 degrees c
- Tissue is placed in the melted wax, heat causes xylene to evaporate
- paraffin cools and hardens
Sectioning
Slicing hardenned tissue using a microtime to thin it out so light can pass through
cuts 6 microns for LM and .06 for EM (thinner b/c is an electron beam, aka more powerful) know this
Mounting
tissue sections are mounted on on glass slide (LM) or wire grid (EM)
Staining
Staining tissue with hematoxylin (H) in water and Eosin (E)
HE stain for LM
What are the steps for section staining?
- Dissolve paraffin wax with xylene
- re-hydrate section through a series of alcohols stained with H-stain in water
- de-hydrate section through a series of alchols stained with eosin (E-stain)
What is the most common dyes used for tissue staining?
Combination of H&E
What is the ultimate goal for preparation of tissues for microscopic examination?
Enable transmission of a light or election beam through tissue to produce a visible image
Enable transmission of a light or election beam through tissue to produce a visible image it requires slicing of tissue into thin sectios and attaching the sections to what?
A glass slide (LM) or wire grid (EM)
Eosin is and ____ dye and carries a net ____ charge
acidic, negative
Eosin reacts with a ____ charge and you get a ____ colored stain
positve, pink
Hematoxylin is an overall ____ dye and carries and net ___ charge.
basic, positive
Hematoxylin reacts with a ____ charge and you get a ____ colored stain
negative, blue
Hematoxylin stains what types of organelles?
Nucleus/ nucleic acids
Ex: RER bc ribosomes have a lot of nucleic acids
rna rich organelles (ribosomes + rER)
RER is in the ___ ____ so it stains____?
basal membrane, blue
basals
The lumen and secreatory vesicles are part of the ____ group so it stains ____.
apical, pink
After fixation of cells and tissues, the ____ of their components differs from living tissues.
chemical composition
After fixation, what remains in the tissue sample?
Large insoluble molecules or macromolecular complexes
fat gets washed out
What is the only organelle that you can see on a light level?
Nucleus
you can get an impression of other organelles with staining
Often a viewed ____ unit is also a ____ unit.
structural, functional
Some large and small molecules are generally lost during tissue prep due to what?
the use of organic solvents during tissue preparation
We can characterize a cell by ____ and ____.
nucleus, shape
You are looking at an ____ through fixation and staining.
artifact
____ is an example of distortion.
Shrinkage
What are artifacts of prep?
Collection of distortions caused by prep procedures (shrinkage, artificial spaces, loss of molecules, etc.)
Shrinkage causes
fixation, dehydration (in ethanol), heating (in melted paraffin during clearing)
What are artificial spaces?
Holes b/w cells + ec components. This is caused by shrinkage and loss of molecules due to chemicals used in tissue prep.
Some ____ components don’t stain well with HE so they stain w/ Heavy metals
neuro
What you see through eyepiece is an accumulation of ____ and ____ of eyepiece
objective, magnification
What is the maximal resolving power of the light microscope?
0.2 microns
What is the magnification for LM?
1000 to 1,500 x
What is resolving power?
How far apart they have to be to resolve as 2 points/separate objects
(the smallest t distance b /w 2 particles at which they can be seen as sep objects)
resolving to a certain light is based in the WL of light compared to the WL of beam
Microscopy resolution has to do picture ____ and they closer they get together the ____ it is to resolve them as 2 points.
clarity, harder
quality of image depends on the resolving power.
What is confocal microscopy used for?
Immunifluorescece and uses laser light. The beam is finer so you get finer resolution.
What is the resolving power for EM?
0.1 nanometers, but in practice 3nm
has very high resolution
The high resoolving power (resolution) for EM allows for magnification up to ____ times for isolated molecules and for very thin tissue sections ____.
400,000, 120,000
True or False, EM has a fluorescent screen where you see fluorescene and not light
TRUE
For EM, the beam passes through a condenser that is made of ____ to focus beams.
magnets
Which microscope is used to see atoms?
Ultra voltage TEM
For TDM, it passes electron beam ____ specimen.
through
For SCM, it ____ beam off specimen.
bounce
What are specialized techniques used to localize specific subtances/structures in cells and tissue sections?
Enzyme histochemistry, immunocytochemistry, and autoradiography
What is the light microsope composed of?
Light source, condenser lens, objective lens, occular lens, and mechanical stage for supporting specimen.
What distinguishes TEM vs LM?
It has the same componenets but it uses an electron beans as an “illuminating” beam, magnets for lens, and a fluorescent screen to visualize the structure.
