Electron Microscopy Lectures Flashcards
Why do we use electron as a probe?
- better resolution
(1) High energy electrons have short wavelength that allows us to observe nanoscale features in samples (resolution limit formula)
(2) Electrons interact strongly with matter (good contrast of what you can see)
(3) Easy to produce high brightness electron beams.
(4) Electron beam can be manipulated using electron magnetic field, similar to how optical lenses focus and direct light.
TEM (transmission electron microscopy)
- better resolution
- Specific EM where electrons pass through ultra-thin sample to reveal internal structures at high resolution.
- uses electrons instead of photons to form image (work in vacuum; magnetic lenses instead of glass)
- Ion = electrons = destructive, could be radiation images, so there needs to be sample preservation.
- TEM needs to be large (complex support systems such as vacuum pumps/electromagnetic lenses - vacuum prevents electron scattering and allows for a clear image)
What are requirements for sample preparation for EM?
- Immobilize sample
- Electron resistant
- good contrast
- as intact as possible
What are some dilemmas that you may encounter when preparing your sample for EM?
(1) Biological sample is aqueous/hydrated, soft, light elements (C/O/H/S/P), large. Need to transfer to a SOLID STATE, preserved the structures as a function of the living states. (any treatment may change the specimen).
What are the steps for classic sample prep for EM?
1) Fixation (immobilize sample)
2) Dehydration (to prevent evaporation in EM vacuum - replace H20 with solvent)
- can be done in ethanol or acetone
- may have consequences for ultrastructure preservation & immunohistochemistry.
3) Embedding (solidify sample, in plastic)
- sample embedded into epoxy resin (no water, polymerization by heat) solidifying into block. enables ultra thin sectioning and heavy metal staining for imaging.
OR
(2) Lowicryls (polar/non-polar), can be some water, photopolymerization by UV, freeze substitution.
- the plastic is only soluble in solvent-acetone.
- harden sample for cutting without distorting it.
4) Thin sectioning (<100 nm)
- blocks in TRAPEZOID, keeps track/order of earlier vs late cuts, longer edge = first section, clear sequence when analyzing samples.
5) Staining (contrast enhancement)
6) TEM (imaging)
- thin sections -> heavy metal salts (solution) to enhance scattering contrast of specimens by increasing mass density differences of various components of tissues and cells, increase scattering of electrons.
- form complexes -> increase density.
- conventional double staining - uranyl acetate & lead citrate/ osmium & tannic acid.
What is used for the fixation step in LM vs EM?
(1) LM:
- Formaldehyde (small size, fast tissue penetration, quick cross-linking)
(2) EM:
- Glutaraldehyde (stronger fixation, better ultrastructure preservation)
Fixation stops biological processes in cell as quick as can. It immobilizes (crosslinking in sample) and preservers cell morphology wither with chemicals or by rapid freezing (cryo-fixation).
Immunogold Electron Microscopy
- to localize molecules in cells, tissues at high resolution.
(1) sections incubated with primary Ab.
(2) Secondary Abs conjugated to 5-20nm gold particles. - high scattering easily seen in TEM. amplification purposes. cost/ease of experiment. different sizes of gold beads.
- immuno-EM provides much better resolution compared to immunofluoresence.
How is EM used as a tool in cell biology?
(1) Morphology/change in morphology caused by various diseases.
Example: Nemaline Myopathy; dark rods (composed of aggregates of z bands)
(2) Immungold labeling used to localize proteins at high resolution. (can see if protein is mislocalized)
- such as seeing the accumulation of intraneuronal B-amyloid 42 peptides (Alzheimer’s Disease)
(3) EM observation is a fast diagnostic tool.
- viruses have characteristic morphology, can identify them easily.
- identify infectors agents (SARS coronavirus, HIV, Ebolavirus)
- SARS: when cells are infected with coronavirus, virus binds to membrane and cilia, viral entry through cilia. Infection leads to the loss of cilia, impaired mucus clearance, triggers pathway affecting cilia function.
In TEM, How can we retrieve 3D information from 2D images? (2D images can be misleading)
(1) Serial Sections
- sample cut into ultrathin slices and imaged sequentially.
- trapezoid shape keeps track of slices.
- limitation: z-axis resolution depends on section thickness, X/Y resolution can be much higher. (resolution is limited by section thickness)
- provides 3D without major changes to the process.
(2) Tomography
- overcomes z-resolution limits of serial sections
- provides better 3D detail of structures within a single section
- multiple images taken from different angles of same object, then combined to reconstruct 3D model.
- captures accurate 3D details without needing multiple sections.
What are some potential problems with conventional EM samples preparation?
(1) Fixation
- slow (sec -> minutes)
- conformation changes of protein
- permeability change of membranes
- osmotic effect leads to dimension alteration
- loss of diffusible ions and small molecules
- masking of antigens
(2) Dehydration
- shrinkage
- conformation change of proteins
- loss of lipids
(3) Embedding
- mechanical effects
- shrinking due to polymerization
- loss of lipids
(4) thin sectioning
- compression, knife marks
(5) Staining
- artifacts (precipitation of heavy metals)
(6) TEM
- Interpretation mistakes
What are some tech advances to look at the sample in more native conditions?
- CryoEM
- Observation of sample frozen (hydrated)
BUT placing it in liquid, fridge would damage it by forming ice crystals (destroy sample)
So
We use VITRIFYING WATER (process of freezing sample without forming ice crystals) - turning a sample into glass-like, non-crystaline solid.
- biological samples quickly frozen by plunging into liquid ethane at liquid nitrogen temperature.
- improves ultrastructure preservation (have to free really fast, at least 10,000C/S)
Why do we use liquid ethane and not liquid nitrogen when freezing our sample in CryoEM?
Because of the Leidenfrost Effect!!!
- when liquid touches a much hotter surface it forms a gas layer that prevents direct contact (why when we dip out hand in liquid nitrogen it wont freeze, cause liquid nitrogen turns into gas, creating insulating layer)
- we use liquid ethane instead cause nitrogen is too cold (-196C) cause leidenfrost effect and prevents proper freezing.
Liquid ethane is cold enough to vitrify but warm enough to avoid this effect. - Water is around 20 degrees, needs rapid cooling without forming ice crystals, which liquid ethane allows.
In terms of the density of the sample, what is the difference between classical EM and Cryo-EM?
(1) Classical EM:
- reflects what is being stained rather than the full density of the sample.
- Resin: poor structure, low resolution, best contrast.
(2) CyroEM:
- provides more accurate density map.
- can see thin cell area/cellular organelles.
- best ultrastructure preservation.
- best resolution.
BUT low contrast.
How can we fix the issue of low contrast in CyroEM? Can we find a way to see atomic details so we can understand how cells function?
- can do single particle EM.
- the requirement is that you need purified protein complexes, not organelles or cells.
- works by imagining many copies of the same protein in different orientations (assume all proteins have the same structure)
(1) Take many 2D images from diff. angles.
(2) Assign & classify images based on their orientation.
(3) use back-projection to reconstruct 3D model of protein, by combining 2D images.
What is the Cryo-EM workflow?
1) Sample prep-purify.
2) Sample freezing (vitrify) & loading.
3) Automated image acquisition.
4) Data storage and handling.
5) 3D reconstruction/image processing.
Single Particle Analysis
1) particle picking
- identify location of protein complexes (particles) in image.
- manual picking vs automated.
2) 2D classification
- 2D images groups based on their appearance (orientation, conformation, composition)
- used to discard wrongly picked particles/damaged particles.
Then, you do 3D alignment and reconstruction.
What are the (+) of Cryo-EM structure determination?
- easy sample prep.
- molecules in closer-to-native.
- requires only small amounts of sample.
- more forgiving on sample purity.
- give information on sample dynamics.
What is an example of scientist doing structural analysis of a protein using cryo-EM (single particle)?
- SARS-Cov2-spike protein
- express spike protein using a plasmid, purify, vitrify, analyze in diff orientation -> 3D image. then could design vaccines.
- highlights key mutation (N50IY), enhances ability to bind to human ACE2 receptor, important for infection.
- omicron spreads easily, weakened Ab binding (mutations reduce vaccine effectiveness), stronger ACE2 binding (still infect cells but other mutations compensate)
knowing this helped researches update treatments.
