In Vivo Monitoring

Question 1

What are fluorophores?

Answer 1

A functional group within a fluorescent molecule, it absorbance and re-emits energy and reports on molecular environment.

Question 2

What structure to fluorophores have?

Answer 2

An aromatic or conjugated structure.

Question 3

What are the classes of fluorescent molecules?

Answer 3

Xanthene derivatives, cyanine derivatives, pyrene derivatives and derivative of naphthalene, coumarin, oxadiazole and acridine.

Question 4

What are the possible filters for fluorescence?

Answer 4

630 nm BandPass filter
Standard Long Pass Filters (520nm)
Standard Short Pass Filters (575nm)
Dichroic filters

Question 5

What are dichroic filters used for?

Answer 5

Used to control light through microscopes and flow cytometers and cut off enables light to be reflected or transmitted.

Question 6

Why is fluorescence used?

Answer 6

As without it cells and tissue are predominantly transparent under a light microscope and there is little contrast, no detail and no specificity. Allows you to use antibody technology to accurately locate proteins and structures of interest, provides good contrast, bright signal on a black background. Enables a number of targets to be visualised in a single experiment, interactions and dynamics.

Question 7

What are biological applications of fluorescence?

Answer 7

Fluorescent microscopy, flow cytometery and real time PCR

Question 8

How is fluorescence used in biological applications?

Answer 8

They are conjugated to antibodies and modified nucleic acids via functional groups: amino, carboxyl and thiol groups- immunofluorescence.

Question 9

What are the two types of immunofluorescence?

Answer 9

Direct and indirect

Question 10

What are the advantages of using immunofluorescence?

Answer 10

Sensitivity, enhancement or weak signal, multicolour applications and flexible.

Question 11

What are the disadvantages of using immunofluorescence?

Answer 11

Samples need to be fixed (dead) and often have holes punched in them through permeabilisation with detergent to allow antibody access to intracellular antigen.

Question 12

How are the disadvantages of immunofluorescence overcome?

Answer 12

Using fluorescence proteins.

Question 13

Who won the Nobel prize for chemistry in 2008 for the discovery and development of GFP technology?

Answer 13

Shimomura, Chalfie and Tsien

Question 14

How are protein dynamics studies in live cells?

Answer 14

GFP is fused to specific targeting sequences or to full length proteins.

Question 15

What variations of GFP are there?

Answer 15

Emerald, CFP, Cerulean, BFP, EBFP2, YFP, Venus and mCitirine. Also red variants; mRFP1, DsRed, JRed, mKate, Plum, mCherry, mStrawberry and mTomato.

Question 16

What are the limitations multicolour imaging fluorescent protein combinations?

Answer 16

Quantum yields of the different fluorescent proteins, folding characteristics/ half-life of the different FPs “blinking” and low extinction coefficients resulting in low brightness.

Question 17

How do you control the expression of GFP?

Answer 17

Place GFP under the control of a specific promoter to ascertain the location and activity of the gene of interest.

Question 18

What does FRET stand for?

Answer 18

Fluorescence, resonance energy transfer

Question 19

What are disadvantages of fluorescent proteins?

Answer 19

Size
Artefacts
Mis-targetting
Over expression
Cell toxicity
pH sensitive
Tagging proteins only – what about RNA and DNA?

Question 20

What are fluorescent molecules?

Answer 20

Characterised by their ability to absorb short wavelength light and emit at a longer wavelength.