Lecture 3

Question 1

If you don’t have a confocal microscope how can you get a confocal quality image using a regular fluorescent microscope?

Answer 1

Deconvolve it!

Question 2

What is deconvolution?

Answer 2

Deconvolution uses software that is calibrated to your microscope system to mathematically move out of focused light back in focus.

Question 3

How do you calibrate deconvolution into your microscope?

Answer 3

To calibrate it to your microscope you image fluorescent beads as a control. Because you know the size of the beads, you can easily get that out of focus light back in focus.

Question 4

How does Fluorescence recovery after photobleaching (FRAP) work?

Answer 4

Using fluorescently tagged proteins (Eg. EGFP-Actin) A small regions is hit with a laser for a prolonged period of time until there is no fluorescence remaining. After time that fluorescent area recovers. The time tells us the turnover (how fast it comes back) time of those proteins you were looking at.

Question 5

What can Fluorescent Resonance Energy transfer (FRET) be used for?

Answer 5

We can take advtange of the emitted light from a fluorophore to excite a nearby fluorophore to see if the 2 molecules are closely associated using this method.

Question 6

How does Fluorescent Resonance Energy transfer (FRET) work?

Answer 6

Use 2 fluorescent proteins.
Excite the first one.
It emits light that will excite the second one.
Then collect the light from the second one only.
Eg. CFP excited at 433/emits at 475nm.
Yfp excites at 475/emits at 530.

Question 7

What are we always limited by?

Answer 7

Abbe limit of 0.2um.

Question 8

How can we get past the optical resolution limit?

Answer 8

Use Super-Resolution Microscopy

Question 9

What is structured Illumination (SIM)?

Answer 9

A grid is placed between the light path and the camera.
3 pictures are taken with the grid in 3 different positions.
Only the light that is in the same positions is kept, leaving a sharp image with little out of focused light.
Can get to 100 nm resolution (not bad.
Very slow because you need 3 pictures for every plane.

Question 10

What is Stimulated Emission Depletion (STED)?

Answer 10

It works just like the point-scanning confocal, but has a Ring (AKA. a depletion beam) around the laser point.
This makes the excited area much smaller.
It takes all of the point, puts them together to build an image.
Resolution ~30nm.

Question 11

What is the problem with Stimulated Emission Depletion (STED)?

Answer 11

Very slow!

Question 12

What is Photo-Activated Localization Miscroscopy (PALM)?

Answer 12

Thousands of images are collected each with only a few molecules of a photo-activable fluorescnet protein (Eg. PA-GFP) excited.
These molecules are black to start off with, then fluorescence when activated.
The centre of the excited photons will give the location of the PA-GFP molecule itself. That is what is documented.

Question 13

What is the problem with Photo-Activated Localization Miscroscopy (PALM)?

Answer 13

It is very very very very slow!!
There is 10nm resolution (essentially perfect resolution).

Question 14

What is Stochastic Optical Resolution Microscopy (STORM)?

Answer 14

It is nearly identical to PALM, and it is really slow.
There is 20nm resolution (when it was invented), now down to ~5nm (phenomenal)!
Instead of using PA-GFP-type of molecules (so starting black and activating the colour)… like with PALM.
Lasers are used to excite a photoswitchable flurochrome to a dark state and turn on the fluorescence with 1 wavelength and off with another.
The centre positions of each molecular image is plotted base off of the photons emitted.

Question 15

What does Stochastic mean?

Answer 15

Random

Question 16

What is Super-resolution Microscopy (lattice light sheet)?

Answer 16

It uses a sheet of light to illuminate 1 entire plane of the sample being imaged in that same plane.
All planes are imaged very quickly (up to 1000 images/second).
Resolution ~ 150nm (so it is super-resolution, but there are better microscopes for resolving structures).
This enables live 3D super-resolution imaging in any direction you want.

Question 17

How thick or thin are the tissue sections for imaging?

Answer 17

Most imaging happens with either transparent cells or thin tissue sections (5-10um) or transparent organisms.

Question 18

What happens with thick samples?

Answer 18

Like a whole brain that you want to see, the distribution of the blood suplly or trace a single neutron.
OR
A kidney and you want to see a protein in a specific cell in the natural context of the brain (not ripped out of the organ).
What can you do?
TISSUE CLEARING

Question 19

What is tissue clearing?

Answer 19

Why would you want to clear the tissue?
It can give you the 3D arrangment of whtver you’re looking at within huge volumes (100um-several cm)
Couldn’t you just use confocal or mulitphoton imaging?
Yes, but image quality gets progressively worse as the thickness increases. Also for thick samples, you have to use infrared wavelengths of light.
By clearing a sample you get rid of much of the matter that will generate out of focused light (the lipids are the problem) and can use light in the visible spectrum.

Question 20

How do you “clear” a sample?

Answer 20

Example: Hydrogel embedding
Embed the samples in a hydrogel
Remove lipids in 8% SDS
Immerse in a clearing solution (FocusClear, 80% glyverol, there are many others)
End-up with a clear sample
Depending on the technique, it can take hours to months
Can cause the tissue to shrink or swell
Can easily be used for immunolocalization as there is no membrane hampering the permeability of the probes being used

Question 21

What is electron microscopy?

Answer 21

Electron microscopy uses a high quality velocity beam of electrons to shoot at the same.
Resolution of 0.0005nm (the size of an atom) due to the short wavelength of the electrons
40,000 times better than a light microscope!
Usually the resolution is ~0.1nm for biological samples; 2000X better than a light microscope; 300X better than super-resolution
All under an ultrahigh vacuum because air can absorb electrons.
Electrons are contained within an electric field
This field condenses the beam of electron through the sample

Question 22

What are the limitations to electron microscopy?

Answer 22

Can’t do live imaging

Question 23

What are the 3 types of electron microscopy?

Answer 23

1) Transmission EM (and ImmunoEM)
2) Scanning EM
3) 3D EM tomography

Question 24

What is Transmission EM?

Answer 24

Trasmission electron microscopt lets you see structures within cells.
Samples are fixed by cross-linking the proteins into position using fixatives (Paraformaldehyde/ Glutaraldehyde).
Embedded in a resin
Sectioned into ~70nm sections.
Stained with heavy metals

Question 25

What is ImmunoElectron microscopy?

Answer 25

Using immunoEM we can detect antigens on structures at the ultrastructual level.
Gold particles are used to identify their lcotion because they are electron dense.
Dual labeling is also possible using gold particles of different diameters (e.g. 5nm and 15nm particles)

Question 26

What is Scanning electron microscopy?

Answer 26

See the surfaces of samples
Samples are coated with metals
The image is actually the secondary electrons being released from the coated metal particles that are detected using a cathode tube (or now a computer)
Resolution ~10nm

Question 27

What is CryoEM?

Answer 27

No fixation or staining needed
Samples are frozen with liquid nitrogen
So, the samples are viewed in their native state
Hundreds of images get averaged to generate the final image.
This technique got the Nobel prize in chemistry in 2017.

Question 28

How does CryoEM Tomography work?

Answer 28

Here the sample is rotated on a platform so that larger samples can be imaged from many different angles.
Ther esulting series of images are computationally reconstructed to see their 3D organization.

Question 29

What are some problems with electron microscopy?

Answer 29

If you want to label a protein, then you need an antibody.
You can’t correlate something seen on a fluroescent microscope with an electron micrograph from the same sample.
How can we fix this?
Use miniSOG!

Question 30

What is miniSOG?

Answer 30

A genetic tag for proteins
Labels proteins green (like GFP)
It makes a 106 amino acid protein (NOTE: that is ~1/2 size of GFP)

Question 31

What can miniSOG catalyze?

Answer 31

Can catalyze a chemical (diaminobenzidine) through miniSOG’s generation of using oxygen, into a reaction product that can be easily stained using osmium.
This makes an electron dense reaction product that can be used as a label.