Fluorescence Microscopy Flashcards Preview

Core Lectures > Fluorescence Microscopy > Flashcards

Flashcards in Fluorescence Microscopy Deck (46):
1

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

Bacteria
Sperm cells
Blood cells

2

When was the first simple microscope invented and who by?

By Van Leeuwenhoek around 1668

3

When was the first compound microscope invented and who by?

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

4

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

Robert Hooke

5

What are the types of microscope aberrations?

Axial chromatic aberration
Spherical aberration

6

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

Neighbouring molecule

7

Define Stokes shift

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

8

What can we use the Stokes shift for?

To separate the excitation and emission light in a fluorescence microscope

9

What do immunolabels tag?

Epitopes

10

What does FISH stand for?

Fluorescence in situ hybridisation

11

When was GFP discovered?

1962

12

Who discovered GFP?

Shimomura et al.

13

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

2008

14

What species was GFP isolated from?

The jellyfish Aequoria victoria

15

What does GFP stand for?

Green fluorescent protein

16

Who first cloned GFP?

Prasher et al.

17

In what year was GFP first cloned?

1992

18

Where can GFP be fused to a protein?

At either ends of the protein or within the protein

19

GFP folds in all organisms, true or false?

True

20

What external factors are needed for GFP fluorescence?

No external factors needed except light

21

What proteins can be tagged by GFP?

Most proteins

22

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

Martin Chalfie

23

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

False

24

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

A large molecule
Might change dynamics, localisation, interactions or function of the protein

25

How many amino acids are there in GFP?

229

26

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

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

27

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

Roger Tsien

28

Define photobleaching

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

29

What does FRAP stand for?

Fluorescence recovery after photobleaching

30

Define FRAP

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

31

Why study FRAP in cells?

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

32

How are samples viewed using a wide field microscope?

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

33

Describe how a confocal laser scanning microscope works

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

34

What is a voxel?

A 3D pixel

35

What are the advantages of light sheet microscopy?

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

36

What is the Rayleigh criterion associated with?

Resolution

37

How can we improve resolution?

Try to use a shorter wavelength
Using electron microscope
Use FRET

38

What does FRET stand for?

Forster resonance energy transfer

39

What wavelength do electrons have?

~5pm

40

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

'Super resolution microscopes' or nanoscopes

41

Give an example of a super resolution microscopes

STED

42

What does STED stand for?

Stimulated emission depletion microscopy

43

How does STED work?

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

44

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

2014
Betzig, Hell, Moerner

45

What are the advantages of fluorescence microscopy?

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

46

What are the disadvantages of fluorescence microscopy?

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