Lecture 4 Flashcards
What can we see in LM?
-Tissue organization
-Only a few organelles
Why can’t we build an LM to see very small things?
Resolution: Resolution is the smallest distance between two point that can still be distinguished
Low resolution vs High resolution
Low: pixelated
High: looks human
What does a larger resoltuion mean?
More pixelated image
Limit of the resolution of the LM using an eye objective?
200 nm
Resolution equation?
R = 0.61λ/nsinθ
How to improve resolution?
Use something with a smaller wavelength
T/F: High energy = short wavelengths
Yes
Why do we use electrons as probes?
- High energy electrons have short wavelngth = better resolution
- ELectrons interact strongly with matter to give good contrast
- Easy to produce high brightness electron beams
- Can focus electron beams at specific point using magnetic field
First electron microscope
1931
How is EM different from LM?
-Electron source(EM)/Light source(LM)
-Condenser for both
-imaging lens(EM)/Objective lense(LM)
-Magnification lenses in both
-Detectors for EM
Why are elctrons bad?
Cause radiation damage which makes sample preservation harder
Why is the EM large?
Electrons interact very strongly with matter, so if you have a low vacuum in the electron microscope the electron would collide with everything in the air. There needs to be a really high vacuum inside so that the electrons actually hit the sample rather than the air
Requirements of a sample for EM?
- Must be resistant to high vacuum
- Immoblized
- Reistant to the elctron beam
- Thin (<300nm)
- Good contrast
How are samples prior to preparation for EM?
- Aqueous
- Soft
3, Made up of C, O, H, S, P these elements do not refract light properly = low contrast - Large
Name sample prep steps in order?
- Fixation
- Dehydration
- Embedding
- Thin sectioning
- Staining
- TEM
Goal of fixation?
Stop biological processes
Crosslink the sample
Preserve cell morphology
How do we fix cells?
Glutaraldehyde/formaldehyde
(usually glutaraldehyde)
or by cryo-fixation
Goal of dehydration
Remove water from the specimen
Resin is insoluble in water
How do we dehydrate the sample?
Using Ethanol or Acetone to 100%
Can affect ultrastructure
Goal of embedding?
Harden the sample for cutting without distoring it
How do we embedd the sample?
- Epoxy resins (Epon Spurr, LR white)
-High temp - Lowicryl
-Low temp embedding
How do we section the sample?
-Ultra-microtome
-Cut in trapezoid to know which sections were cut first
Goal of staining?
Introduce contrast
How do we stain a sample using heavy metal salts?
-Heavy metal salts increase mass density differences of various components(increases scattering of electrons)
-More dense structures get more contrast
How do we stain a sample using conventional double staining?
-First uranyl acetate then lead citrate
- Or osmium then tannic acid
Why do carbohydrates appear white on EM?
Normally the stain used has low specificity for carbohydrates
Stain is bound look dark vs no stain is white?
Yes
What can we see using TEM?
- Tissue organization
- Cell organization
- Organelle morphology
- Big protein complexes(nuclear pores, cytoskeleton)
Immunogold EM
- Section is incubated with primary antibody
- Secondary antibodies are linked with a gold nanoparticle
Disadvantage of immunogold EM?
The antigen of interest has to be at the surface of the section
Advantage of EM immunogold vs Fluorescent LM
Immunogold:
-Easily seen due to high resolution of EM
-EM allows us to also see the surrounding environment of the protein whereas in LM we would need to create more antibodies to see the surrounding
How can we label multiple proteins using immunogold?
-Use secondary antibodies coupled to different size of gold
Immunogold Labelling limitations
-Proteins might not be recognizied by the antibodies due to processing
Tokayasu’s cryo-sections
Processing is done at cold temperature (nothing denature), sample is kept partially hydrated, embedding is done once the sample has been cut
This is better for immunogold
TEM as a diagnostic tool
-EM can detect how morphology of cells change in disease
-Immunogold can be used to see protein mis-localization
-Viruses can be identifies
