Electron Microscopy 1 Flashcards
1mm =
100um = 1,000,000nm = 10,000,00 angstroms
Mammalian cell sizes
7-30um
Describe lm
From ancient times, man has wanted to see things far smaller than could be perceived with the naked eye.
Invention of optical microscope:
Use of glass to magnify things dates at least to the Romans with the invention of glass in the first century.
In 1665 Hooke published Micrographia, a book describing observations made with microscopes and telescopes, as well as some original work in biology.
It is widely believed that the Dutch spectacle makers, Zacharias Jansen and his father Hans were responsible for making the first compound microscope in the late 16th century.
What can we see with lm
Histology - tissue organization
Phase contrast, differential interference contrast
Why can’t we build a lm that can see v small things
Bc resolution
What is reolsution
Smallest distance between 2 points that can still be dinstinguished
- less resolution = becomes single dot
Resolution formula
R = 0.61lamda / n sin theta
Want smaller value - if high res = small value
Describe theta of resolution
Half of angular width of cone of rays collected by objective lens from typical pint in specimen
What is n in formula
Refractive index of medium separating specimen from objective and condenser lens
Increase = makes resolution better, but cannot increase n much
Describe lambda in formula
Wavelength of light used
High energy= short wavelengths = high spatial resolution
Why use electrons as probe
Resolution = high ernergy with short wavelengths = can observe nanoscale features in samples
Electrons interact strongly with matter = gives v good contrast of what we can see
Easy to produce high brightness electron beams - cheap and easy
Electron beam can be manipulated using electron magnetic field - can focus it
Em history
- Potential of using electrons for imaging was understood the beginning of the 19th century.
- The first electron microscope was built in 1931 by Ernst Ruska and Max Knoll at the Berlin Technische Hochschule.
- The initial instrument was capable of magnifying objects by only 400 times, demonstrating the principles of an electron microscope.
- Two years later, Ruska constructed an electron microscope that exceeded the resolution possible with an optical microscope
- It was greatly developed through the 1950s and has allowed great advances in the natural sciences and physics.
In north America - in Canada, rca microscopes
Name components of tem
Electron source
Sample illumination - condenser lenses
Imaging lens - objective
Magnification and projections - intermediate projector lens
Detectors - eyes/camer
What does tem use
Electrons instead of photons - works in vacuum, magnetic lenses instead of glass
Why is tem good
Improves resolution
Why is tem bad
Electrons are destructive = cause damage
Radiation damage, sample preservation
Why tem so big
Bc when e- hits sample = give off x rays - so need to protect from this
Also bc e- interacts strong with matter- so if low vacuum = e- would collide with everything in air
= need it to be empty inside = vacuum
What is sample prep requirements for ‘em
Immobilize sample
Electron resistant
Good contrast
As intact as possible - so represents cell well
What are biological samples chemically made out of
Proteins(~chainsofaminoacids).
DNA/RNA/nucleotides(~chainsofnucleotides).
Sugar
Lipids
Water(70%ofthecells)
COHNP
Describe biological samples characteristics
Aq-hydrated
Soft
Light elements - cohhsp - will not defract electrons properly
Large
= need to be trasnfer into a solid state - which preserves functions and structure of living state
Describe em characteristic
High vacuum
Sensitive to vibration
Electron beam
Limited penetration
What are reqs for sample prep
Resistant to high vacuum
Immobilized
Resistant to e- beam
Thin = <300nm for tem
Good contrast
Name classical sample prep steps
Fixation
Dehydration
Embedding
Thin sectioning
Staining
Tem
Describe fixation
Formaldehyde - bc much smaller so penetrates faster than glutaldehyde (although its better fixer)
Formaldehyde = lm
Glutadldheye = em
Immobilize sample
Solvent dissolve biological matter
Describe dehydration
Replace water with solvent bc would evaporate and also for embedding = cannot have water since plastic solubilized in solvent not watere
Solvent dissolve biological matter
Describe embedding
Solidify sample - embed in block
Plastic only
Need solid specimen
Describe thin sectioning
<100nm
Describe staining
Can do heavy metal
Contrast enhancement
Then tem and imaging
Describe ex of sample prep
Glutaraldehyde fixation - perfusion
Odium tetroxide= fix and that preserves cell membrane
Propylene oxide - 3x10 mins
Liquid resin
Polymerizaion - oven 60degres - depends on expoxy
Polymerized resin block = can cut it
What are goals of fixation
Stop biological processes in cell as quick as possible
Immobilize - cross link sample
Preserve cell morphology
What are methods of fixation
With chemicals = glutaraldehyde/formaldehyde
By rapid freezing - cryo fixation
What are goals of dehydration
Remove water entirely bc hard to cut
Resin soluble in solvent - not water
Resins = polymers which can be hardened with heat or uv but this reaction inhibited by water
What are methods of dehydration
Ethanol or acetone to 100% to remove moisture
Process may have consequences for ultarstructure preservation and immunocytochemistry
What are goals of resin embedding
Harden sample for cutting without distorting it
Describe expoxy method of resin embedding
Resins - Epon, spurr, lr white
Water immiscible
Polymerization by heat-12-24hrs at 60dgrees - bad preservation of epitopes tho
Describe lowicryls resin embedding method
Polar - hydrophilic k4m
Or a non polar - hydrophobic hm20
Photopolymerized by uv - 360nm - better for preserving antigens for igg labelling
K4m = specimens may be kept in partially hydrated state - polymerization up to 5% water in block
Freeze substitution - low temp embedding - below 50degrees
Describe sectioning or ultramicrotomy
Thin
Tweezers pick up grid
diamond knife edge - resin embedded specimen block will be sliced - can adjust thickness then = Sections in water
Delicate
Trapezoid shape = bc want to know which one early slice vs later slice - this way can tell if have long vertex
Goal of staining
Introduce contrast for sample
Methods of staining
Thin sections usually stained with solutions of heavy metal salts = enhance scattering of Contrast of specimens by increasing mass density differences of various components of tissues and cells - this increasing scattering of electrons
Metal ions of staining solutions form complexes with certain components of cells= increase their density
Often - staining has little chemical specificity but contrast of components such as ribosomes and membranes is increased relative to their surroundings= Not very specific - certain stains better for diff things
Conventional double staining -first in uranyl acetate followed by lead citrate
Also odium and tannic acid
Describe staining flow chart
Floating em grid contains the section upside down on top a drop of water and stain solution
Em grid = with thin sections —> wash h2o —blot/dry—> 2.5% uranyl acetate —blot/dry—> wash h2o —blot/dry—> Storage/imaging (can store for years)
New era in cell biology - what can we see using tem
TIssue organization at high res
Cell organization
Cell morphology
Big protein complexes = npcs, cilia - csk
What is weakness of ‘em
How do we know what we are looking at
What are we looking at
See dots mainly = important to label proteins and stain them so we know what we seeing
Describe lm immunofluorescence
Antigen - primary antibody first then secondary binds primary
Use detergent to permeabilzie cell = can be specific to Pam and not nuclear membrane or whatever
Describe immunogold em - gen
Very hard
Label section
Localize molecules in cells, tissues at high res
Secondary antibody has gold nano particle - linked to primary antibody
Describe immunogold em - staining process
Sections incubated with primary antibody- generated to recognize specific protein
Secondary antibodies - conjugated to 5-20nm golf Particles = high scattering = easily seen in tem, amplification purposes, cost/ease of exp
What is advantage of em immunogold vs fluorescent lm
See in high res exactly where protein fo interest is
Also an see surrounding environment = see whole cell contents
Resolution Great
How to label multiple proteins sue immunogold
Use secondary antibodies coupled with diff sizes gold beads = see size
Also weakness bc hard to see diff sizes- can only label 3-4 diff sizes
Describe immunogold limitations
Ability of proteins to be recognized by antibodies can be affected by processes
Classical processing not optimal for preserving immunogeneicity = to keep antigens = not easy since heat in oven
Describe immunogold limitations - alternative methods
Cryo sectioning = most processing done at cold temp
Sample kept partially hydrated
Embedding done once sample has been cut
Cryo sectioning and embedding - antigen not destroyed
Em as tool in cell bio - morphology
See morphology and change in morphology causes by Dsieases
Ex = nemaline myopathy
The dark rod-shaped subsarcolemmal inclusions seen here in this electron micrograph are known as nemaline rods. The “rods” are composed of aggregates of Z bands= can see looks wrong compared to wild type muscle cell
Em as tool in cell bio - immunogold
Can see if proteins mislocalized - not v well tho
Ex = accumulation of amyloid in alzeihmers
= can see protein not distributed properly
Em as tool in cell bio - em observation
Fast diagnostic tool = vrisues have their characteristic morphology that allows to identify them easily
= see sars, hiv, Ebola , Covid on cilia of lung
Describe case study - sars cov2
Saw it on cilia = thought it maybe enters through cilia
What effect of covid on cilia
Conscious = sars cov2 = triggers pathway that leads to cilia loss and impairs mucus clearance = see morphology purely
Why immunogold bad
Antigen = has to be on surface - antibody cannot go in
