Microscopy

Question 1

What is the function of the microscopy?

Answer 1

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

Question 2

What are the parts of the microscope?

Answer 2

• Detector (PMT, CCD)
• Objective (+/- immersion medium)
• Specimen (cover glass)
• Light conditioning system
  
  • > Kohler illumination
  • > Phase ring
  • > Wollaston prism and polarizers
  • > Filter cubes (for fluorescence)
• Light source (Halogen, XBO,…)

Question 3

Why is a light source needed?

Answer 3

A light source is needed but can be modified depending on the complexity of the microscope

Question 4

Describe the light path of a light source

Answer 4

1. Light reaches the specimen and enables us to see the sample
2. Goes through the lenses and then we can see the image
3. The computer generates the image

Question 5

Why is a cover glass of the same thickness used?

Answer 5

To maintain standard across all the microscopes with a cover glass of the same thickness

Question 6

What is ‘The Box’?

Answer 6

It is a box used to prevent focus instability.

It controls CO2 and temp

Question 7

Why is an incubator box used?

Answer 7

It is used to stop small changes in temperature leading to thermal extension or contraction of the microscope stand, stage, and objective. Also combined with a precision air heater ensures the temp of a specimen and microscope remain constant.

Question 8

Why is there a controller on the box?

Answer 8

Allows adjusting airflow and the %CO2. - also, guide the gas stream through a bottle of water in order to diminish the loss of humidity

Question 9

Why is there an airtight tabletop?

Answer 9

It encloses the live cell culture device

Question 10

What is the issue if it takes too long to observe something under a microscope?

Answer 10

May be problems with maintaining the stability and the liability of the sample such as CO2 and temp.

Question 11

What is the issue if you capture too fast with a microscope?

Answer 11

Need to distinguish the sample, it can be hard to capture, need to capture the exact sample and not its surroundings. Can compromise the resolution when taking a picture of the fast-moving image

Question 12

What is the triangle of frustration?

Answer 12

• Temporal resolution
• Sensitivity
• Spatial resolution
  A particular point or moving around

Question 13

Why is it important to adjust spatial resolution?

Answer 13

• Used to capture a fast-moving image: sacrifice the resolution to capture the movement
• Use the triangle and sacrifice one thing or another

Question 14

What are magnifiers?

Answer 14

A high-quality piece of glass in a vertical way. Allows to see things bigger

Question 15

What is magnification?

Answer 15

The number represents how big the objective can magnify

Question 16

What are the applications of the different magnifiers?

Answer 16

Modification of the light for different applications can make sure it is suitable for the specific use. All microscopes are set in a way that they can be used to their best ability.

Question 17

Why are emersion mediums needed?

Answer 17

• As not all objective lens are ready to see the sample just through air = needs to be immersed in a different medium such as water or oil -cannot function without the emersion.

Question 18

What does the aperture of the objective determine?

Answer 18

The resolution on the microscopy. The higer the numerical aperture, the better the resolution power of the objectives. The resolution does not cause magnification.

Question 19

What is the difference between resolution and magnification?

Answer 19

Resolution refers to the detail of the picture. It can have the same magnification between two pictures but have different numerical aperture. The higher numerical aperture means more detail. Numerical aperture is linked to resolution but not to magnification.

Question 20

Structure of a light microscopy

Answer 20

• Can have bright field and a fluorescence mode
• Has a light source, objectives and sample plane
• As well as, Polarizer, Wollaston Prism, Condenser, Phase Ring

Question 21

Describe how absorption and emission can cause flourescence

Answer 21

1. Energy is generated and a wavelength is exicted. (The molecules that have been excited meet another wavelength)
2. The wavelength excites the molecule and reaches a peak.
3. Then, this starts to lose energy and the different wavelength is emitted.
4. Light/Fluorescence is emitted

Question 22

What is the Stoke’s Shfit?

Answer 22

Due to energy loss the emitted light is shifted to longer wavelength relative to the excitation light. The environment causes the cycle to break known as stokes shift.

Question 23

Define the stokes shift

Answer 23

The difference between wavelength of the molecule of interest and the wavelength of the emitted wavelength

Question 24

What is photobleaching of the fluorochromes?

Answer 24

• Due to high intensity illumination.
• The fluorophores might lose permanently their ability to emit light.
• The gain of energy cannot be released anymore as an emission fluorescence- through time to see is decreased.

Question 25

Why is photobleaching done?

Answer 25

- To work with reduced excitation light intensities/grey filters: - > Use shorter exposure times - > Higher gain settings - > Longer intervals during the time lapse studies - > Use anti-bleach in you mounting media

Question 26

What are fluorescent proteins and why are they used?

Answer 26

Fluorescent proteins can be fused with other proteins and introduced into cells via transfection. It allows live studies of fluorescent tags in living cells/organism.

Question 27

Give an example of a fluorescent protein

Answer 27

GFP - found naturally in light-producing cells of cnidarians

Question 28

When are fluorescent proteins used?

Answer 28

1. Plasmid construction where the GFP protein is inserted into the ES cells through electroporation 2. This allows for the selection of stable ESC clones by G418. 3. The green fluorescence can help identify alive vs dead.

Question 29

What are haemocytes?

Answer 29

- They display contact inhibition of locomotion - The cells migrate and are isolated enough to be able to establish short context between them to know where they are. - Long microtubule filament where actin filament can converge. It would have been impossible to see without the fluorophores

Question 30

What can be used to create a time lapse?

Answer 30

A fluoroescence microscopy

Question 31

Why is a laser used in a confocal fluorescence microscopy?

Answer 31

Laser instead of a light source. The light released from the sample enters a small circle and enables there to be more detail.

Question 32

Compare widefield and confocal fluorescence microscopy

Answer 32

- There is higher z-resolution and reduced out of focus blur made = confocal pictures are crisper and clearer. - Only a small volume can be visualised by confocal microscope at once but bigger volumes need time consuming sampling and image reassembling - Can do tissue and cellular localisation - Can also make 3D reconstruction models