Fluorescence Microscopy

Question 1

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

Answer 1

Bacteria
Sperm cells
Blood cells

Question 2

When was the first simple microscope invented and who by?

Answer 2

By Van Leeuwenhoek around 1668

Question 3

When was the first compound microscope invented and who by?

Answer 3

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

Question 4

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

Answer 4

Robert Hooke

Question 5

What are the types of microscope aberrations?

Answer 5

Axial chromatic aberration

Spherical aberration

Question 6

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

Answer 6

Neighbouring molecule

Question 7

Define Stokes shift

Answer 7

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

Question 8

What can we use the Stokes shift for?

Answer 8

To separate the excitation and emission light in a fluorescence microscope

Question 9

What do immunolabels tag?

Answer 9

Epitopes

Question 10

What does FISH stand for?

Answer 10

Fluorescence in situ hybridisation

Question 11

When was GFP discovered?

Answer 11

1962

Question 12

Who discovered GFP?

Answer 12

Shimomura et al.

Question 13

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

Answer 13

2008

Question 14

What species was GFP isolated from?

Answer 14

The jellyfish Aequoria victoria

Question 15

What does GFP stand for?

Answer 15

Green fluorescent protein

Question 16

Who first cloned GFP?

Answer 16

Prasher et al.

Question 17

In what year was GFP first cloned?

Answer 17

1992

Question 18

Where can GFP be fused to a protein?

Answer 18

At either ends of the protein or within the protein

Question 19

GFP folds in all organisms, true or false?

Answer 19

True

Question 20

What external factors are needed for GFP fluorescence?

Answer 20

No external factors needed except light

Question 21

What proteins can be tagged by GFP?

Answer 21

Most proteins

Question 22

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

Answer 22

Martin Chalfie

Question 23

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

Answer 23

False

Question 24

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

Answer 24

A large molecule

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

Question 25

How many amino acids are there in GFP?

Answer 25

229

Question 26

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

Answer 26

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

Question 27

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

Answer 27

Roger Tsien

Question 28

Define photobleaching

Answer 28

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

Question 29

What does FRAP stand for?

Answer 29

Fluorescence recovery after photobleaching

Question 30

Define FRAP

Answer 30

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

Question 31

Why study FRAP in cells?

Answer 31

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

Question 32

How are samples viewed using a wide field microscope?

Answer 32

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

Question 33

Describe how a confocal laser scanning microscope works

Answer 33

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

Question 34

What is a voxel?

Answer 34

A 3D pixel

Question 35

What are the advantages of light sheet microscopy?

Answer 35

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

Question 36

What is the Rayleigh criterion associated with?

Answer 36

Resolution

Question 37

How can we improve resolution?

Answer 37

Try to use a shorter wavelength Using electron microscope Use FRET

Question 38

What does FRET stand for?

Answer 38

Forster resonance energy transfer

Question 39

What wavelength do electrons have?

Answer 39

~5pm

Question 40

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

Answer 40

'Super resolution microscopes' or nanoscopes

Question 41

Give an example of a super resolution microscopes

Answer 41

STED

Question 42

What does STED stand for?

Answer 42

Stimulated emission depletion microscopy

Question 43

How does STED work?

Answer 43

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

Question 44

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

Answer 44

2014 | Betzig, Hell, Moerner

Question 45

What are the advantages of fluorescence microscopy?

Answer 45

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

Question 46

What are the disadvantages of fluorescence microscopy?

Answer 46

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