Electron Microscopy

What is the order of size for biological objects?

Biggest to smallest:

• mammalian cells

• nucleus

• bacteria/mitochondria

• virus

• ribosomes

• proteins

• membranes

• water molecules

• atom

What is the resolution of the light microscope?

200nm (visible light)

anything past this 200nm uses super resolution and other techniques need to be used

What is resolution?

resolution (d) is the smaller distance between two points that they can still be distinguished as two separate points

• nowadays there are more pixilations in screens which is why the images look so much more smooth

• with better resolution you can see finer details

How can the equation be manipulated to improve the resolution? what is the limit?

• resolution = 0.61 lambda / n sin theta

• theta maximum can be 180 degrees → meaning that sin theta can be a maximum of 1

• to improve resolution we can:

  • increase n and sin theta

  • decrease wavelength

• best resolution from a light microscope:

  • wavelength = 400-700nm

  • n (oil) = 1.4

  • sin theta max = 1

  • 0.61×400nm / (1.4×1) =174.286 Nm 174nm

  • virus cannot be seen using a light microscope

• however with an electron microscope we can get a better resolution because can use really short wavelengths (you can theoretically see an atom)

Why do we use electron as a probe?

• electrons interact strongly with matter

• easy to produce high brightness electron beams

• the electron can be manipulated using electron magnetic field because it has a charge

Why don’t we have an x-ray electron microscope?

it can’t be manipulated using a magnetic field because it doesn’t have a charge

Who built the first electron microscope and when?

it was built in 1931 by Ernst Ruska and Max Knoll

What are the components of the TEM?

• you need a “light” source

• condenser lens to illuminate the sample

• imagining lens to focus the light

• magnification and projections (intermediate and projector lens)

• detectors

• EM uses electrons instead of photons to form an image (work in a vacuum; magnetic lenses instead of glass

  • this improves resolution but electrons are destructive and cause radiation damage to the sample)

How do we prepare the sample for EM?

• immobilize the sample using a fixative (formaldehyde)

• electron resistant

• stain to get good contrast

• as intact as possible

What are the constituents of a biological sample?

• proteins

• DNA/RNA/nucleotides

• sugar

• lipids

• water (70% of the cells)

• COHNP (99% are COHN)

Why isn’t carbon and hydrogen good for electron microscopy?

its a low atomic number and does deflect electrons well so it won’t give good resolution

What else must you do to prepare the microscope?

• must have high vacuum

• sensitive to vibration

• electron beam

• limited penetration

What must you do them to prepare the sample?

• resistant to high vacuum

• immobilized

• resistant to electron beam

• thin

• good contrast

What are the steps for classical sample prep?

1. fix

2. dehydrate

3. embed

4. thin sectioning (using diamond knife)

5. stain

6. TEM

What is the goal of fixation and how do we fix the sample?

the goal:

• stop the biological process in the cell as quick as possible

• immobilize the sample

• preserve cell morphology

methods:

• with chemicals (gluteraldehyde/formaldehyde)

  • use gluteraldehyde in EM allows for more efficient cross linking

• by rapid freezing (cry-fixation)

What is the goal of dehydration and how do we dehydrate the sample?

the goal:

• remove all the water because it is difficult to cut if water is present

• resin is soluble in solvent not water

• resin can be hardened but this is inhibited by water so it needs to be removed

methods:

• specimens can be dehydrated with ethanol or acetone to 100% to remove moisture

• may have consequences for ultrastructure preservation and immunocytochemistry (loss of antigens in this process)

What is the goal of embedding and how do we embed the sample?

the goal:

• harden the sample for cutting so that the sample doesn’t get distorted

the method:

• use epoxy resin that can be hardened using heat

How does Ultra microtomy work?

uses water well????

What is the goal of staining and how do we stain the sample?

the goal:

• introduce contrast for the sample

methods:

• thin sections are stain with solutions of heavy metal salts to enhance the scattering contrast of specimens by increasing the mass density differences of various components of the tissues and cells (increases scattering of electrons

• conventional double staining → first in uranyl acetate followed by lead citrate

• osmium and tannic acid can also be used

What is the process for staining?

What can we see using TEM?

• tissue organization at high resolution (nerve tissue and skeletal muscle)

• cell organization (pancreas cell and plant cell)

• organelle morphology (the RER, nuclear envelope, Golgi)

• big protein complexes (nuclear pore complex, cytoskeleton cilia,

How can we identify the protein we are looking at in the cell?

using immunofluorescence

• indirect

• use a primary antibody that recognizes the antigen

• use a secondary antibody that recognizes the primary antibody

• couple with fluorescence

must also make sure that the antibodies can pass through the membrane because the things we are labelling are usually inside the cell → we use detergents which make the membrane permeable (create holes allowing for penetration

What is immunogold labelling?

What is its advantage of immunogold compared to fluorescent LM?

How do we label multiple proteins using immunogold?

use different gold particle of a different size

What are the limitations of immunogold?

ability of the proteins to be recognized by the antibodies can be affected by processing → classical processing is not optimal for preserving immunogenicity

What is an alternative method to immunogold?

Tokayasu’s cryo sections:

• processing done at cold temperatures

• sample kept partially hydrated

• embedding is done after the sample is cut

How is EM used as a tool in cell biology?

• morphology and change in morphology

• Immunogold labeling continues to a tool frequently and routinely used to localize proteins at high resolution

• EM observation is a fast diagnostic tool

Why are 2D images not representative of a 3D object?

because if you only look at it from one direction you don’t get the full picture → looking at it from one direction shows one image but it may look like something else if you rotate it in another direction

How can we retrieve 3D information from 2D images?

• serial sections

• tomography

What is wrong with traditional TEM images being 2D?

• A flat image can misrepresent the true shape of a structure.

• Sample preparation (resin embedding) can distort tissues.

• Resolution is often limited by how samples are prepared, not the microscope itself

What is serial sectioning?

the specimens is cut into many consecutive thin sections and imaged in order to obtain the 3D view

Why is the specimens cut into trapezoid shape?

to keep track of the top and bottom of the specimens so that when you go to put the individual 2D images together you create the proper 3D image

How does serial sectioning work?

1. using novel ultra-thin sectioning technique, very thin slices of the specimen are cut (50-100nm thick)

2. each slide is images seperately

3. images are stacked to reconstruct a 3D structure

*stacking resolution is limited by the techniques used to cut the sample

What are the advantages of serial sectioning?

• allows visualization of 3D organization

• similar to a confocal Z-stack in light microscopy → confocal light microscope takes many images at different focal planes along the z-axis → provides sharp, high resolution images of thin optical sections inside a thick sample

Why doesn’t the normal light microscope allow you to get Z section like confocal?

confocal has a special construction where there’s a pinhole to block out-of-focus light

limitations of serial sectioning?

• resolution is limited by section thickness

• alignment can be difficult

What is tomography?

technique used to create 3D image of the inside of an object by combining many 2D images (slices) taken from different angles or depths

What are the advantages of tomography?

• serial sections cant achieve very high Z-resolution but this can

• it gives better 3D detail inside thicker sections

How does tomography work?

1. collect lots of images of the object from different viewpoints or layers by rotating the object

2. computer mathematically combines those images (back projections)

3. result is a 3D reconstructed image of the internal structure

*uses thicker sections

Who won a noble prize in 1979 for X ray computed tomography?

Cormack and Hounsfield

Who won a Nobel prize in 1982 for applying tomography principle in TEM?

Klug

What are the general steps of EM prep?

1. Fix

2. dehydrate

3. embed

4. section

5. stain

6. imaging

Whare some problems with conventional EM sample preparation?

• fixation is slow

• shrinkage and deformation

• loss of lipids

• conformation change of proteins

• permeability change of membranes

• staining artifcaitos

• mechanical damage

• misinterpretation of structures

What happens in classical processing?

• biological sample is 70% water

• we totally remove the water and we cook it → 60 degrees for 48 hours

  • this retains certain features but not all components are preserved → ie. fresh apricot vs dry apricot

What is Cryo-EM?

sample is observed in a frozen state → imaged → then viewed with an electron microscope

avoids crystallization of the sample or the need for chemical fixation

Why can’t we just freeze the sample by putting it in the freezer?

crystal ice is formed destroying the structure → must use another technique called vitrification

What is vitrification? What about specifically in the context of Cryo-EM?

is the transformation of a substance into a glass (a non crystalline solid)

the sample is in a middle stable state → the ling you are looking at is in a liquid state but it behaves like a solid

WITH CRYO-EM?

• vitrification of biological samples in solution on the EM grid by plunging a small volume of sample quickly into liquid ethane at liquid nitrogen temperature

How does Cryo-EM work?

1. a tiny drop of sample is placed onto a metal grid

2. the grid is flash frozen extremely fast in liquid ethane

3. water becomes vitreous ice instead of ice crystals

4. grid is loaded into a cryo-EM and left at -180 degrees

5. beam of electrons passes through the sample

6. using a software the images are aligned

7. reconstruction occurs via tomography

What are the benefits of Cryo-EM? How does it improve ultrastructure preservation?

• instead of using chemicals to fix we can freeze it really fast

• ice crystals are damaging

• if we freeze it fast ice crystals don’t form but rather amorphous ice does

• if we observe the sample at a cold temperature crystals still don’t form

• sample is frozen hydrated and in its native state

Why do we freeze in liquid ethane at the temperature of liquid nitrogen and not directly in liquid nitrogen?

Liquid ethane enables faster heat transfer than liquid nitrogen, avoiding gas insulation and allowing vitrification of water without ice crystal formation

• leidenfrost effect: the formation of a gas barrier between a hot surface and a boiling liquid if the temperature difference is great enough → the grid is very hot for the boiling temperature of liquid nitrogen

• When a warm grid is plunged into liquid nitrogen, nitrogen rapidly boils and forms a gas layer around the grid, which insulates it and slows heat transfer. This prevents rapid freezing and allows ice crystals to form. Liquid ethane, cooled by liquid nitrogen, does not form this insulating gas barrier and has higher heat transfer efficiency, allowing ultra-rapid freezing and vitrification of the sample.

What are the benefits of cryo-EM?

• Structures preserved close to native state.

• Less distortion and shrinkage

However it is very vulnerable to radiation because its a real cell (can only allow for so much electron exposure)

What is cryo-EM tomography?

technique that lets scientists see the 3D structure of intact cells or cellular components in a near-native, frozen state using an electron microscope.

It combines:
Cryo-EM → frozen preservation and high resolution
Tomography → 3D reconstruction from many images

Difference between cryo and resin?

Cryo:

• best ultrastructure preservation

• best resolution

• low contrast image

resin:

• poor structure preservation

• low resolution

• best contrast

what can we do at higher resolution?

• understand how the cell functions at the molecular level (protein level)

• understand how proteins function

What is single particle cryo-EM?

• it is a type of Cryo-EM

• only deals with purified protein complexes

• Images many identical copies of the same protein or complex.

• Thousands–millions of particles are aligned and averaged.

• Averaging removes noise → very high resolution (often near-atomic)

how does single particle cryo-EM work?

1. vitrify the ribosome

2. put in electron microscope and take pictures

3. group the ribosome into groups of similar ribosomes

4. many identical particles are averaged to reduce noise

Which is higher resolution SP cry-EM or cryo-ET?

Single particle cryo-EM is higher resolution than cryo-ET.

Describe the Single Particle Analysis cryo-EM workflow (cry-EM sample preparation)

1. purify the protein and vitrify it

2. bring to microscope and collect images over night

3. pick particles from images

4. Classify the images into similar views → group based on similarities and appearance (ie. orientation, conformation, composition)

5. process the images to get a reconstruction of the protein

What are the benefits of cryo-EM?

• easy sample preparation

• molecules in closer-to-native state

• requires small amount of sample (5 microlitres used during freezing)

• gives info on sample dynamics

What happened with the Omicron virus?

there is a mutation originated from the other covid variant so the antibodies in the vaccine are inefficient

Which 3 cryo-EM developers won the noble prize in chemistry in 2017?