Lecture 2

Question 1

What was used in microscopes instead of fire?

Answer 1

Light bulbs were used, then very bright mercury arc lamps

Question 2

What are mercury arc lamps?

Answer 2

Mercury arc lamps use electricity to excite mercury vapour to generate light

Question 3

What did objective lenses do?

Answer 3

Objective lenses with different magnifications on a turret enables scientists to quickly change the magnification of samples

Question 4

What is the “Limit of Resolution”?

Answer 4

The limit of resolution is always 0.2 um (200nm). This means that you can only resolve/identify structures that are 0.2 um or bigger. The limit of resolution to as the Abbe limit. This is because Ernest Abbe calculated it. But did he actually figure out the formula? Probably not. It is reported that the formula was derived 61 years prior from the mathematician Joseph-Louis Lagrange. That didn’t really matter as Abbe became a partner with carl Zeiss and a co-owner of Zeiss.

Question 5

What are dichroic mirrors?

Answer 5

Allow light of a certain wavelength to pass through, but reflects all other wavelengths

Question 6

What is magnification?

Answer 6

Objective lenses (closest to the samples) usually max-out at 100X magnification. The projection lens or eyepiece lens is 10X magnified. The total possible magnification is 1000X.

Question 7

What is bright field microscopy?

Answer 7

Bright field is used on unstained samples.
The most basic way to see a sample.
Uses few lenses and 1 light source. Light is concentrated onto the sample using a condenser lens (to focus light on specimen).
Light hits the sample (radiates at all different directions).
Magnified by the objective lens and projection lens.
Hits the detector (your eye or a camera).
Little amounts of detail can be seen.

Question 8

What is phase-contrast microscopy?

Answer 8

Phase-contrast microscopy is used on unstained samples.
It takes advantage of the “refractive index” of the material. Refractice index is a number (from a calculation) that describes how light passes through a sample.
It produces an image with dark and light areas based on the refractive index of those zones of the sample or cell.

Question 9

What does refractive index have to do with speed of light?

Answer 9

Light moves slower in areas with a high refractive index.
The light is refractive (bent) when it passes through the object.
These microscopes use an “annular diaphragm” that only allows some of the light to pass through it.
That light hits the sample.
Then only some of that light passes through the “phase plate”.
To ultimately go to your eye or camera.

Question 10

What is differential interference contrast (DIC)?

Answer 10

It is used on unstained samples.
It breaks light into 2 perpendicular components.
Then passes them through the sample.
The light is then recombined.
The interference pattern is then observed.
Useful for seeing small details in cells.
Uses the refractive index of the sample and the surrounding material to generate what looks like shadows.

Question 11

What is fluorescence microscopy?

Answer 11

The most versatile technique.
Can identify the location of specific molecules.
Can use those molecules to label structures.

Question 12

How does fluoresence microscopy work?

Answer 12

Bright light
Lenses
Dichroic mirrors (mirrors that allow certain wavelenghts of light to pass, but not others)
Detection devices

Question 13

When does fluorescence occur?

Answer 13

Fluorescence happens when a molecule absorbs light at a specific wavelength (the excitation wavelength) and emits it at a longer wavelength (the emission wavelength).
When labelled multiple things in a simple sample you don’t want the emission spectra from 2 colours to overlap beause then you won’t be able to easily tell which is which.
Yes, there are ways to “unmix” them using software, but they are not easy.

Question 14

What is an example of fluorescence microscopy?

Answer 14

Light at all wavelenghts is generated, but only a certain wavelength passes through the excitation filter

Question 15

What is a dichroic mirror?

Answer 15

A dichroic mirror allows certain (selected) wavelenghts of light through, all others bounce off.
The light that reflects from the dichroic mirror excited the chemical flurochrome attached to the molecule to be visualized on the sample.
The fluorochrome emits light at a different wavelength that then passes through the same dichroic mirror ultimately to your detector (eye or camera).

Question 16

What are some fluoroscence techniques?

Answer 16

Immunofluorescence (Direct or Indirect)
Molecular Probes
Fluorescent Proteins
Antibodies

Question 17

What is immunofluorescence (or molecular probes?

Answer 17

Direct immunofluorescence is when there is no secondary step to it. Indirect is when it involves primary and secondary. Primary antibodies recognize microtubules and flurochrome-conjugated secondary antibodies (indirect).

Question 18

What is fluorescent proteins (GFP)?

Answer 18

Genetically formed fluorescent proteins that can be tagged to other proteins for visualization.

Question 19

What happens when you take an image you focus on 1 plane of your sample, but excited the entire sample?

Answer 19

This generated a lot of out of focus light (going to distort a lot of light).

Question 20

How can you get rid of out of focus light?

Answer 20

1) Use computers to remove it
2) Change your light source

Question 21

What is phototoxicity and why is it a problem for live imaging?

Answer 21

Phototoxicity is when high powered light sources (arc-lamps or lasers) are used they generate free radicals in the cells which ultimately kill the cell.

Question 22

How can we try to fix phototoxicity?

Answer 22

1) Lower the intensity of the light hitting the sample
2) Decrease the exposure time
3) Put “thing” in the light path

Question 23

What is Confocal (Point-scanning, AKA. Laser Scanning confocal microscope [LSCM])?

Answer 23

Uses lasers to excite the sample
Takes images from ONLY 1 specific focal plane… (so then there is very little out of focus light)
Collects images from the entire sample (1 focal plane at a time)
These can be re-assembled using computes into 3D reconstructions
Uses a “pinhole” to further ensure that our of focus light is elimated
Uses a photomultiplier tube to build the image from the area of the sample being illuminated from the scanning laser

Question 24

What are the problems of Confocal (Point-scanning, AKA. Laser Scanning confocal microscope [LSCM]?

Answer 24

1) It’s slow…. really slow. So if you want to live image a sub-cellular event, you might miss it
2) Because it’s so slow and the laser is so intense the fluorescent molecule can bleach out (or the fluorescent protein can be destroyed)

Question 25

What is confocal (spinning disk)?

Answer 25

Splits the laser light into many many lenses and pinholes (20,000 of them).
This enables the scanning of the entire sample in milliseconds.
This decreases the exposure time and the intensity of the laser beam hitting the sample.
Commonly used for live imaging

Question 26

Where does light from a confocal go?

Answer 26

Light from a confocal must blast through the top of the sample to reach the focal plane of interest.
This causes un-needed damage to that region of the sample.

Question 27

How can we avoid un-needed damage to the sample?

Answer 27

Using Two-photon/Multiphoton microscopy

Question 28

What is Two-photon/Multiphoton microscopy?

Answer 28

Uses multiple lasers at half of the intensity (or 1/3 or 1/4 the intensity… then you need more lasers) to excited the fluorescent molecule.
Will results in the same emission from the fluorescent molecule.
Generates less phototoxicity (removes light).
Ideal for live imaging
Including intravial live imaging

Question 29

What is the problem with Two-photon/Multiphoton microscopy?

Answer 29

Lasers are very expensive!