Part 2A: Background

Question 1

Describe fluorescence imaging

Answer 1

Detection of light emitted from a sample upon irradiation

Question 2

Advantages of fluorescence imaging

Answer 2

• Better contrast than offered by reflection or transmission alone
• Can achieve different localisation, including with several emissive species

Question 3

Outline the two key types of fluorescence microscopy

Answer 3

Epifluorescence
* Focussing excitation onto sample through an objective lens; only that which is reflected hits the detector
Confocal microscopy
* Excitation light passed through a pinhole focussed through objective lens onto a tiny area on the focal plane (z axis)
-> Emitted light undergoes the same process
-> Entire image can be scanned onto xy plane and repeated after shifting the focal plane

Question 4

Pixel, Voxel

Answer 4

• Pixel: Spot in xy plane
• Voxel: Reconstituted 3D point

Question 5

Confocal microscopy: 2 advantages and 2 disadvantages

Answer 5

+ Very high signal:noise ratio
+ High resolution
- Limited tissue penetration (<1mm)
- Abbe’s diffraction limit

Question 6

Give 5 properties of useful fluorophores

Answer 6

• Large stokes shift (appreciable difference between signal and autofluorescence; Abs-Em)
• Long lifetime; can utilise time-resolved microscopy
• Excited and emitted light must not damage tissue (i.e. high energy UV)
• Localisable via properties like lipophicity or polarity
• Biocompatible (Non-toxic, stable in biological media)

Question 7

How do fluorophores operate?

Answer 7

• Excitation of e- through absorption of light
• Energy loss through vibrational levels and electronic levels
• Reemission of light of longer wavelength (lower energy)
• Most organic fluorophores involve fluorescence from singlet states (short lifetimes)

Question 8

Give 4 essential properties of luminophores:

Answer 8

• Stability and Solubility (in aqueous buffers, growth media)
• Toxicity (No photo-toxicity upon irradiation)
• Readily uptaken into cell (High lipophilicity, cationic charge, mass <500 Da)
• Localisation (preferential to a certain organelle or easily adapted by bioconjugation) -> tunable

Question 9

Typical commercially available fluorophores, give an example

Answer 9

• Planar, aromatic heterocycles
• Either pi to pi* or n to pi* transitions
• Singlet excited state
• Often available with reactive groups to attach to
  e.g. 5-IAF

Question 10

How do charge transfer fluorophores operate?

Answer 10

• Charge transfer from electron rich to electron poor
• Photo-induced charge transfer leads to a charge-separated excited state

Question 11

What are fluorophores typically responsive to?

Answer 11

• pH responsive e.g. fluorescein
• Potential responsive
• Analyte responsive

Question 12

pH responsive agents:

Answer 12

• Equilibrium between ring-closed form and open, emissive form illustrates a responsive probe
• Allow pH mapping (more acidic areas are less fluorescent)
• Issue in differentiating between areas of low uptake vs acidity

Question 13

Potential responsive agents:

Answer 13

• Agent with a membrane associating unit shows charge transfer excited state, so can assess membrane potential (e.g. in nerve signalling)
• Polarisation makes charge transfer more favourable -> wavelength of excitation red shifts

Question 14

Analyte responsive agents:

Answer 14

• Properties change upon binding to analyte
• Allows detection and potentially concentration detection
• Useful for NO (vasodilation, viagra localisation) -> with amines
  e.g. BODIPY diamines emit at 500nm vs 530 upon NO reaction
• Facilitates ratiometric sensing

Question 15

Ratiometric sensing:

Answer 15

• Different wavelength with and without analyte
• Using ratios to derive concentration of analyte
• Utilises calibration curve

Question 16

What is the most common approach in organelle targetting?

Answer 16

Biological approaches such as Antibody tagging.

Question 17

DNA/RNA targeting:
(3 examples)

Answer 17

• Planar aromatics
• Bind via groove or intercalation (cationic species)
  e.g. DAPI, Hoescht dyes (Minor groove binders)
  e.g. Acridine orange (true intercalator)

Question 18

Mitochondria targeting:

Answer 18

• Lipophilic and cationic to cross mitochondrial membrane
• Accumulate due to mitochondrial membrane potential
• React through benzyl chloride group (Sn2 displacement with thiol from glutathione)
• Produces a very polar adduct which remains, even in daughter cells

Question 19

Membrane targeting:

Answer 19

• Highly lipophilic (C12-C18)
• Attracted by lipophilicity and hydrophobicity

Question 20

Protein tagging: (with 3 examples)

Answer 20

• Dyes with either amine or carboxylate
• e.g. IAF (reasonably selective for thiol residues)
• e.g. DANS-Cl, FITC ( target N-termini)
• Can also target antigen by tagging fluorophore with antibodies