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