Microscopy

Question 1

1. What is microscopy?

Answer 1

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

Question 2

2.What are the ‘eyes’ or ‘camera’ called on a microscope? Give two examples of what type this could be

Answer 2

Detector

PMT or CCD

Question 3

3. What is the magnifier of a microscope called?

Answer 3

The objective

It can + or - the immersion medium

Question 4

4. Where would you find the specimen on a microscope?

Answer 4

Cover glass

Question 5

5. What types of light conditioning systems (modify the light )are there?

Answer 5

• Kohler Illumination
• Phase Ring
• Wollaston Prism and Polarisers
• Filter Cubes (for fluorescence)

Question 6

6. What is the purpose of the light source in a microscope?

Answer 6

(Halogen,XBO): Different ways of conditioning how the light reaches the specimen.

Question 7

7. How many mm’s are a cover glass?

Answer 7

0.17

Question 8

8. What is a cover glass surrounded by?What might this contain?

Answer 8

Cover glass surrounded by embedding medium which might contain anti-bleach agent

Question 9

9. How might small temperature changes affect a microscope?

Answer 9

Small temp changes –> thermal extension/contraction in stand,stage and objective in microscope –> change the plane of focus

Question 10

10. How can we overcome issues of temperature changes in a microscope?

Answer 10

“The Box”

An incubator box combined with precision air heater —> temp of specimen and microscope equilibrated and controlled.

Question 11

11. How do we maintain the CO2 atmosphere?

Answer 11

A controller allows us to adjust air flow and the conc of co2 and an air tight table top encloses the live cell culture device

Question 12

12. How can you use a bottle of water to diminish loss of humidity?

Answer 12

Theres a possibility of guiding the gas stream through a bottle of water–> diminish loss of humidity

Question 13

13. What do experimental timescales depend upon?

Answer 13

The type of organism ( simple or complex)

Movement (fast or slow)

Question 14

14. How long is movement in comparison to development ?

Answer 14

Movement is seconds

Development can be hours/days

Question 15

15. What is an advantage and disadvantage for a longer experimental timescale?

Answer 15

Adv: Fewer Artefacts

Disad: More variation in temp

Question 16

16. What does a short experimental timescale require?

Does it produce artefacts?

Answer 16

Machinery that can detects changes in positions very quickly

Yes, produces artefacts

Question 17

17. What are the three points of the signal transduction :triangle of frustration?

Answer 17

• Temporal Resolution
• Sensitivity
• Spatial Resolution

Question 18

18. Can we have all three simultaneously :
    - Temporal Resolution
    - Spatial Resolution
    - Sensitivity

Answer 18

Nope!

Usually 1 has be sacrificed e.g. to see fast images we have to sacrifice spatial resolution etc.

Question 19

19. What does Spatial resolution mean?

Answer 19

Number of pixels used

So if you use loads of smaller pixels = High spatial resolution (clearer image)

Question 20

20. What does temporal resolution?

Answer 20

This is more to do with time, so if you want a high temporal resolution (a quick image) you usually have a less clear image (lower spatial resolution)

Question 21

21. Fill in the blank:

The longer light has to travel, the ******* the temporal resolution.

Answer 21

Lower

Question 22

22. 
On an objective you will be given the :
Magnification
Application
Coverslip thickness (mm)
Numerical Aperture
Immersion Medium
Working Distance
What do each of these mean?

Answer 22

Magnification = Size
Application = Use
Coverslip thickness (mm) = If its too thick, no magnification
Numerical Aperture = Measure of ability to resolve fine detail 
Immersion Medium = Medium that light can go through ( ie water/oil) . light reflection is diff
Working Distance = Furthest distance it can work from

Question 23

23. How does aperture affect resolution?

Answer 23

The higher the aperture = Higher resolution

Question 24

24. How are histology images produced?

laser capture microdissection

Answer 24

Immunohistochemistry: protein of interest is found by using antibodies (produced by injecting animals with foreign material so they produce antibodies in an immune response).
o We can look at large areas of tissue however we cannot see what the cells are doing in detail.

Question 25

25. What is the difference you would see in images of a phase contrast microscopy of fibroblasts cultured on 1. Intact collagen 2. Denatured collagen

Answer 25

Fibroblasts more closely packed with intact collagen

Question 26

26. What is time lapse microscopy?

Answer 26

Time-lapse microscopy is the method that extends live cell imaging from a single observation in time to the observation of cellular dynamics over long periods of time.

Question 27

27. What is light microscopy?

Answer 27

Used to illuminate and magnify images.

Question 28

28. What are the three different types of light microscopy and briefly describe each one

Answer 28

* Brightfield: dark sample on a bright surface. COLOURFUL * Differential Interference Contrast (has shadow): enhance contrast in unstained, transparent samples. 2D LOOKING * Phase: gaining contrast without staining specimen. CONTRASTED

Question 29

29. Give an example of when you would use time lapse microscopy?

Answer 29

Heart cell differentiation: 1. Differentiation of cardiomyocyte-like cells derived from adipocytes. 2.Cell migration

Question 30

30. How is an electron microscope different to a light microscope?

Answer 30

Similar structure to a light microscope except electron source (instead of light source) and a camera (instead of an eye).

Question 31

31. Why would you get a darker image in electron microscopy?

Answer 31

Image is seen when electron go through the specimen or are reflected back, it is more difficult for electrons to go through dense areas - creates a darker image.

Question 32

32. How does Scanning Electron microscopy work?

Answer 32

Well it produces 3D images Instead of sending beam of electrons through the specimen, they are sent at a particular angle and the beam is used to scan the surface image.

Question 33

33. How does fluorescence microscopy work briefly?

Answer 33

Fluorescence allows manipulation as particular wavelengths of light can be used, computers detect the fluorescence reflected from the image.

Question 34

34.How does fluorescence microscopy work in detail?

Answer 34

o Wavelengths of light excites proteins called fluorophores when they absorb light. o As they lose this energy, they emit a particular wavelength of light. o This process is cyclic and could go on forever if in a completely empty environment. o Fluorescence will eventually disappear in a normal sample. o Environment/amount of light can break this cycle in a process known as photobleaching.

Question 35

35. What is Stokes shift?

Answer 35

Difference between wavelength used for excitation and wavelength of emission

Question 36

36. What happens at a smaller wavelength, excitation or or emission

Answer 36

Excitation

Question 37

37. What is Photobleaching?

Answer 37

High intensity illumination bleaches fluorochrome so fluorophores can permanently lose their ability to emit light.

Question 38

38. Whats an example of a Fluorescent protein?

Answer 38

GFP

Question 39

39. In fluorescence microscopy, you can have antibody tags or protein tags. How do ANTIBODY tags work?

Answer 39

AB specific to the protein of interest are added to the sample and a 2nd antibody (with a fluorophore) that recognises the 1st one is added. This allows us to see the protein in colour and localise it.

Question 40

40. In fluorescence microscopy, you can have antibody tags or protein tags. How do PROTEIN tags work?

Answer 40

Generate a plasmid which has the sequence of primary protein linked to sequence of fluorophore. Incorporate the plasmid into the cells, select for cells that have taken the plasmid up and culture these. When cells express the protein, the colour will be seen.

Question 41

41. What is 2 major differences between confocal and Wide-field microscopes?

Answer 41

1. Confocal and Widefield microscopes have similar structures but confocal uses a laser (emits particular light wavelength) as a light source. 2. Confocal blocks most of the laser and only allows it to pass through certain small holes, this allows us to see a thin layer of the cell rather than all of it , Widefield uses more light so we can see the whole cell.

Question 42

42. How does confocal microscopy work?

Answer 42

laser reaches the sample after passing through mirrors, the sample becomes excited and emits a particular wavelength which is enhanced by a photomultiplier (converted into an image).

Question 43

43. List some adv of confocal microscopy?

Answer 43

Crisp/clear images with more detail (due to higher z resolution) higher resolution and reduced out of focus blur

Question 44

44. List some disad of confocal microscopy?

Answer 44

- Only visualises a small volume because bigger volumes need time consuming sampling and image reassembling - Need high powered computers because there is a lot of data to be collected

Question 45

45 . List some uses of confocal microscopy?

Answer 45

• Tissue and Cellular Localisation • Intracellular live imaging e.g. microtubules. • Colocalization e.g. vaccinia virus particles use both microtubules and actin microfilaments. - Confocal microscopy looks at the cell surface. - Vaccinia virus uses actin tails just after crossing the plasma membrane of infected cells. • 3D reconstruction: non-neoplastic and neoplastic (cancerous).