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Core Lectures > Fluorescence Microscopy > Flashcards

Fluorescence Microscopy Flashcards

1
Q

Give examples of some of the things discovered by the simple microscope

A

Bacteria
Sperm cells
Blood cells

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2
Q

When was the first simple microscope invented and who by?

A

By Van Leeuwenhoek around 1668

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3
Q

When was the first compound microscope invented and who by?

A

By Zacharias Janssen, probably with the help of his father in 1595 (considered the first microscope)

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4
Q

Who was the first person to use the word ‘cell’ while describing a piece of cork in 1665?

A

Robert Hooke

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5
Q

What are the types of microscope aberrations?

A

Axial chromatic aberration

Spherical aberration

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6
Q

In fluorescence microscopy, there is some energy loss (heat) sure to conformational changes and collisions with what?

A

Neighbouring molecule

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7
Q

Define Stokes shift

A

Absorption of light with a short wavelength results in the emission of light with a longer wavelength

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8
Q

What can we use the Stokes shift for?

A

To separate the excitation and emission light in a fluorescence microscope

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9
Q

What do immunolabels tag?

A

Epitopes

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10
Q

What does FISH stand for?

A

Fluorescence in situ hybridisation

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11
Q

When was GFP discovered?

A

1962

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12
Q

Who discovered GFP?

A

Shimomura et al.

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13
Q

In what year was the Nobel Prize in Chemistry awarded for the discovery of GFP?

A

2008

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14
Q

What species was GFP isolated from?

A

The jellyfish Aequoria victoria

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15
Q

What does GFP stand for?

A

Green fluorescent protein

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16
Q

Who first cloned GFP?

A

Prasher et al.

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17
Q

In what year was GFP first cloned?

A

1992

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18
Q

Where can GFP be fused to a protein?

A

At either ends of the protein or within the protein

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19
Q

GFP folds in all organisms, true or false?

A

True

20
Q

What external factors are needed for GFP fluorescence?

A

No external factors needed except light

21
Q

What proteins can be tagged by GFP?

A

Most proteins

22
Q

Who first tagged GFP in C. elegans and subsequently won the Nobel Prize in chemistry in 2008?

A

Martin Chalfie

23
Q

GFP cannot be visualised in live cell imaging, true or false?

A

False

24
Q

Is GFP a large or small molecule? What does this mean for its use in protein tagging?

A

A large molecule

Might change dynamics, localisation, interactions or function of the protein

25
Q

How many amino acids are there in GFP?

A

229

26
Q

What additional control measures could you consider in order to control for the size of GFP?

A 
Western blot
Compare N- and C- terminal labelled constructs
Compare with antibody labelling
Use different tag (His-tag, FiAsH-tag)
Protein activity assay
27
Q

Who created different colours of GFP and subsequently won the Nobel Prize in chemistry in 2008?

A

Roger Tsien

28
Q

Define photobleaching

A

The loss of ability to fluoresce due to photon-induced chemical damage and covalent modification as a result of interaction with other molecules

29
Q

What does FRAP stand for?

A

Fluorescence recovery after photobleaching

30
Q

Define FRAP

A

Bleaching a region in the cell using a laser and then following the recovery

31
Q

Why study FRAP in cells?

A

It can give information about the dynamics/binding of a protein

32
Q

How are samples viewed using a wide field microscope?

A

The whole sample is illuminated with light and the whole image can directly be viewed

33
Q

Describe how a confocal laser scanning microscope works

A

Scans pixel-by-pixel
Relatively slow
System only detects light from that part of the sample that is in focus (optical sectioning)

34
Q

What is a voxel?

A

A 3D pixel

35
Q

What are the advantages of light sheet microscopy?

A

Minimal photo toxicity
Fast and sensitive detection using sCMOS camera
Good penetration in scattering tissues
Multi-view acquisition by rotation of the sample

36
Q

What is the Rayleigh criterion associated with?

A

Resolution

37
Q

How can we improve resolution?

A

Try to use a shorter wavelength
Using electron microscope
Use FRET

38
Q

What does FRET stand for?

A

Forster resonance energy transfer

39
Q

What wavelength do electrons have?

A

~5pm

40
Q

In the last decade, many new fluorescence microscope techniques have been developed with an improved resolution up to 10nm, what are these called?

A

‘Super resolution microscopes’ or nanoscopes

41
Q

Give an example of a super resolution microscopes

A

STED

42
Q

What does STED stand for?

A

Stimulated emission depletion microscopy

43
Q

How does STED work?

A

Fluorescence is completely suppressed by a stimulated emission process; the electrons are forced back to the ground state without emitting light

44
Q

When was the Nobel Prize in chemistry awarded for super resolution microscopes and who to?

A

2014

Betzig, Hell, Moerner

45
Q

What are the advantages of fluorescence microscopy?

A

Very sensitive (can detect single molecules)
Highly selective using specific probes
Can be used in vivo
Improved resolution

46
Q

What are the disadvantages of fluorescence microscopy?

A

Usually requires a fluorescent label
Excitation light can be damaging (phototoxicity, bleaching)
Often time consuming
Quantitative imaging is challenging

Core Lectures (13 decks)

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