Microscopy

Question 1

What is microscopy?

Answer 1

Using microscopes to view objects/specimens that are not visible to the naked eye.

Question 2

List the fundamental parts of the microscope from top to bottom.

Answer 2

Detector (PMT, CCD)- in simple microscopes it is our eye, in others a camera or a photomultiplier
Objective (+/- immersion medium)- make the image bigger
Specimen (cover glass)
Light conditioning system- optimises the beam of light in a way to give the best possible image out of the sample
They all aim to either concentrate a beam of light, reduce reflections or filter types of light

Question 3

How is a light microscope specimen prepared?

Answer 3

Sample is usually placed on a slide made of glass surrounded by an embedding medium
Usually covered with an even thinner piece of glass called a cover slip
Some samples are bigger or need to be alive for that we use other methods

Question 4

What is the BOX used for life imaging? Ignore card, even I don’t get it

Answer 4

Preventing focus instability
Even small changes in ambient temperature lead to thermal extension and contraction in the microscope stand, Stage and objective, thereby changing the plane of focus
An incubator box combines with a precision air heater ensures that the temperature of specimen and microscope remains equilibrated and tightly controlled (don’t need to know all of this)

Question 5

How is CO2 maintained in the box?

Answer 5

A controller allows to adjust air flow and the %CO2

there is a possibility of guiding the gas stream through a bottle of water in order to diminish loss of humidity

Question 6

What is the ‘triangle of frustration’ in regards to signal detection?

Answer 6

“The triangle of frustration”
We will need to sacrifice one factor or another
E.g. if you want to follow a long process you will need to sacrifice spatial resolution or sensitivity
- Temporal resolution
- Spatial resolution
- Sensitivity
All detections have their benefits and limitations
What is best, depends on the application requirements

Question 7

How do the corners of the triangle of frustration change an image?

Answer 7

Spatial resolution
- As we reduce the size of the pixels we increase the resolution but that takes longer time
Intensity resolution
- The more information we have the better the intensity resolution but again that takes longer

Question 8

What do the markings on objectives mean?

Answer 8

Magnification
Application
Working distance is the maximum distance that the objective can be from the sample
Numerical aperture (related to resolution) and immersion medium (the type of environment that it can work best)

Question 9

How is resolution determined?

Answer 9

The aperture of the objective determines the resolution
The higher the numerical aperture the better the resolution power of the objective
Resolution ≠ magnification

Question 10

What can you use light microscopy for?

Answer 10

1. Histology
   
   2. Phase contrast- cell morphology
   3. Time-lapse (heart cell differentiation, cell migration

Question 11

What are the pros and cons of histology?

Answer 11

Pros: We can have an overview of the tissue and can identify cells
Cons: We lose the ability to see how these cells interact with each other

Question 12

What is electron microscopy?

Answer 12

Transmission EM
Scanning EM
Despite being more complicated machinery it is still constructed of the same principles
We have the electron source as the source of energy
You have modifiers that modify the electron beam
You have the place for the specimen and magnifiers

Question 13

What is bright field microscopy?

Answer 13

The source of light in this case emitted by a beam which hits the filter cube
That has a series of mirrors that sends the emitted light source to the sample
The sample reacts with the light as it has fluorochromes and that emits another type of fluorescent light that hits the detector

Question 14

What is fluorescence microscopy?

Answer 14

Fluorochromes or fluorophores absorb light at a particular wavelength and when they do they become excited and increase their level of energy
The molecule then loses the energy and goes to a lower level of energy where the fluorophore emits light before going to a ground state and so on in a cycle
This is known as the stokes shift

Question 15

What is photobleaching?

Answer 15

Bleaching of fluorochromes due to high intensity illumination the fluorophores might lose permanently their ability to emit light
-> work with reduced excitation light intensities or grey filters, use shorter exposure times/higher gain settings and longer intervals during time lapse studies; use anti-bleach in your mounting media

Question 16

What are fluorescent proteins?

Answer 16

Fluorescent proteins; i.e. GFP
These proteins are naturally found in light-producing cells of cnidarians
Fluorescent proteins can be fused with other proteins and introduced in cells via transfection
This allows live study of fluorescent tags in living cells/organisms

Question 17

What is the difference between seeing alive and fixed cells fluoresce?

Answer 17

We can attach the fluoresce to antibodies and then you treat the cells of interest with antibodies
But that means that you can only visualise the cells when they are fixed, so not alive
To do this with alive cells involves plasmids that contain the fluorescence gene which is then transfected into the cells so that they can fluoresce

Question 18

What is the difference between confocal and widefield microscopy?

Answer 18

The difference is that in confocal microscopes you have a series of elements that the light goes through before it hits your mirrors that are going to send a particular wavelength to excite your sample
This element is known as a pinhole, a plate that has a very small hole that reduces the reflection of the light allowing a specific wavelength
The sample will react and emit a certain wavelength that goes through the detector pinhole that is captured by a photomultiplier

Question 19

What are the pros and cons of confocal microscopy?

Answer 19

+ Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer

- Only a small volume can be visualised by confocal microscopes at once
- Bigger volumes need time consuming sampling and image reassembling