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Cellular Pathology > Microscopy > Flashcards

Microscopy Flashcards

1
Q
  1. What is microscopy?
A

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

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2
Q

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

A

Detector

PMT or CCD

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3
Q
  1. What is the magnifier of a microscope called?
A

The objective

It can + or - the immersion medium

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

Cover glass

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5
Q
  1. What types of light conditioning systems (modify the light )are there?
A
  • Kohler Illumination
  • Phase Ring
  • Wollaston Prism and Polarisers
  • Filter Cubes (for fluorescence)
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6
Q
  1. What is the purpose of the light source in a microscope?
A

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

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7
Q
  1. How many mm’s are a cover glass?
A

0.17

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

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

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9
Q
  1. How might small temperature changes affect a microscope?
A

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

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

“The Box”

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

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11
Q
  1. How do we maintain the CO2 atmosphere?
A

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

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

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

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13
Q
  1. What do experimental timescales depend upon?
A

The type of organism ( simple or complex)

Movement (fast or slow)

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14
Q
  1. How long is movement in comparison to development ?
A

Movement is seconds

Development can be hours/days

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

Adv: Fewer Artefacts

Disad: More variation in temp

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16
Q
  1. What does a short experimental timescale require?

Does it produce artefacts?

A

Machinery that can detects changes in positions very quickly

Yes, produces artefacts

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17
Q
  1. What are the three points of the signal transduction :triangle of frustration?
A
  • Temporal Resolution
  • Sensitivity
  • Spatial Resolution
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18
Q
  1. Can we have all three simultaneously :
    - Temporal Resolution
    - Spatial Resolution
    - Sensitivity
A

Nope!

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

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19
Q
  1. What does Spatial resolution mean?
A

Number of pixels used

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

20
Q
  1. What does temporal resolution?
A

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)

21
Q
  1. Fill in the blank:

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

A

Lower

22
Q 
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?
A 
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
23
Q
  1. How does aperture affect resolution?
A

The higher the aperture = Higher resolution

24
Q
  1. How are histology images produced?

laser capture microdissection

A

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.

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

Fibroblasts more closely packed with intact collagen

26
Q
  1. What is time lapse microscopy?
A

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.

27
Q
  1. What is light microscopy?
A

Used to illuminate and magnify images.

28
Q
  1. What are the three different types of light microscopy and briefly describe each one
A
  • 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
29
Q
  1. Give an example of when you would use time lapse microscopy?
A

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

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

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

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

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.

32
Q
  1. How does Scanning Electron microscopy work?
A

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.

33
Q
  1. How does fluorescence microscopy work briefly?
A

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

34
Q

34.How does fluorescence microscopy work in detail?

A

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.

35
Q
  1. What is Stokes shift?
A

Difference between wavelength used for excitation and wavelength of emission

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

Excitation

37
Q
  1. What is Photobleaching?
A

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

38
Q
  1. Whats an example of a Fluorescent protein?
A

GFP

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

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.

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

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.

41
Q
  1. What is 2 major differences between confocal and Wide-field microscopes?
A
  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.
42
Q
  1. How does confocal microscopy work?
A

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).

43
Q
  1. List some adv of confocal microscopy?
A

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

44
Q
  1. List some disad of confocal microscopy?
A
  • 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
45
Q

45 . List some uses of confocal microscopy?

A

• 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).

Cellular Pathology (13 decks)

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