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omgmbd > L9: Microscopy > Flashcards

L9: Microscopy Flashcards

1
Q

What are the parts of a microscope?

A 
→detector
→objective
→specimen
→light conditioning system
→light source
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2
Q

What can the light source be?

A

→halogen

→XBO

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3
Q

Describe the light microscopic specimen

A

→cover glass
→sample surrounded by embedding medium
→glass slide

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4
Q

What is the life imaging part of a microscope?

A

→ the box prevents focus instability

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5
Q

How is CO2 atmosphere maintained?

A

→controller allows adjustment of air flow and %CO2

→air tight table top encloses live cell culture

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6
Q

Which cell processes have the shortest experimental time scale?

A

→microtubules

→cytoskeleton

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7
Q

Which cell process have the longest experimental timescales?

A

→differentiation

→development

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8
Q

What is the triangle of frustration?

A

→user have to find the balance between temporal resolution, spatial resolution, and sensitivity

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9
Q

What is temporal resolution?

A

→low exposure time, low pixel number, binning

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10
Q

What is spatial resolution?

A

→high pixel number, no binning

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11
Q

What is sensitivity?

A

→ high exposure time, binning

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12
Q

What are the markings on objectives?

A 
→magnification
→application
→coverslip thickness
→working distance
→immersion medium/numerical aperture
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13
Q

What does the aperture of objective determine?

A

→the resolution

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14
Q

What does a high aperture mean?

A

→The higher the numerical aperture the better the resolution power of the objective.

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15
Q

What are the fundamentals of the light microscope?

A

→Brightfield, DIC, Phase

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16
Q

What are the three stages of light microscopy?

A

1) Histology-
2) Phase contrast
3) Time-lapse

17
Q

How can you locate the protein of interest in histology?

A

→Using Antibodies

18
Q

What is the role of histology in light microscopy?

A

→tissue organisation

19
Q

What is the role of phase contrast in LM?

A

cell morphology

20
Q

What is a problem with using phase contrast in LM?

A

→Can’t determine how changes in morphology went about

21
Q

What is the role of time-lapse in LM?

A

→heart cell differentiation, cell migration

22
Q

What are the two types of EM?

A

Transmission EM

Scanning EM

23
Q

What is the difference between TEM and SEM?

A

→TEM examine the internal structures of a cell, whereas ascanning electron microscope only allows visualization of the surface of a structure.

24
Q

Why can we use electrons to visualise structures?

A

Electrons find it difficult to go through high material areas=darker

25
Q

What are the two types of LM set-up?

A

→bright filed

→fluorescence

26
Q

What are the differences between bright field and fluorescence microscopy?

A

→Fluorescence= source of light(laser beam) goes through filter cube and emits light source to sample,

27
Q

Why do emitted light shift to longer wavelength?

A

→due to loss of energy

28
Q

What is photobleaching?

A

→process whereby fluorophores lose their ability to fluoresce effectively due to high intensity illumination

29
Q

How do you reduce photobleaching?

A

→use shorter exposure

→reduced excitation light intensities

30
Q

What are fluorescent proteins?

A

→GFP

→ naturally found in light producing cells of cnidarians

31
Q

How are live studies of fluorescent tags carried out in cells?

A

→fluorescent proteins can be fused with other proteins and introduced in cells via transfection

32
Q

What is the difference between antibodies and protein fusion for fluorescence?

A

→Antibodies method- cells have to be fixed- when not alive

33
Q

What are the two types of visualising fluoescence?

A

→antibodies

→protein fusion, tag the gene

34
Q

What are the two types of focuses in microscopy?

A

→confocal

→widefield

35
Q

What is a difference between confocal and widefield microscopy?

A

→widefield microscope, the entire focal volume is illuminated, but that creates blur from areas out of focus above and below the image plane

→confocal microscopescans a sample with a focused beam of light, higher resolution

36
Q

Compare confocal and widefield microscopy

A

→confocal has a higher z-resolution and reduced out of focus blur- clearer and crisper
→ only a small volume can be visualised with confocal

37
Q

What do bigger volumes need in order to be viewed by confocal microscope?

A

→need time consuming sampling and image reassembling

38
Q

What can be viewed with intracellular live imaging?

A

→Microtubule dynamics
(GFP-tubulin)
→Vesicle transport through microtubules
(GFP-Kinesin)

39
Q

What are the two types of 3D reconstruction?

A

→non-neoplastic

→neoplastic

omgmbd (29 decks)

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