Part 2: L1, Principles and Basis Flashcards
1
Q
Advantages of fl. imaging
A
- Resolution and sensitivity
- Subcellular level
- More biochemical technique -> detailed
2
Q
Fl. spectroscopy setup
A
- Monochromated using a diffraction grating
- Excitation monochromator perpendicular to emission monochromator where sample re-emits
- Sweep through wavelengths both going in and out -> see which are most effective together -> characterise sample
3
Q
Advantages of confocal microscopy (industry standard):
A
- High sensitivity (nm)
- Low cost
- Specific staining and multi-channel detection
- Responsive; informs on environment
4
Q
Essential properties of lumophores for fluorescence imaging applications:
A
- Stability and solubility
- Toxicity
- Uptake -> Water solubility
- Localisation
5
Q
Structure of phospholipid membrane
A
- Charged head groups on outside
- Nonpolar tails (lipidic)
6
Q
Rules of thumb for lumophore design
A
- Cationic
- Lipophilic
- Mass <500 Da
- Biocompatible, soluble, non-toxic (heavy metals; release of metals)
7
Q
Issues in Fl. imaging:
A
- Background emission (autofl.)
- Self-quenching
- Photobleaching (biochemical loss of agent, typical example is formation of ROS)
- Biocompatability, solubility, toxicity
- Transport-membrane permeability/active uptake
- Tissue damage/penetration
8
Q
Self quenching
A
- Close in space to another dye molecule in case of low stokes shift
- Transfer between molecules loses efficiency (re-absorption by neighbouring molecules loses intensity)
- No overlap if large stokes shift
9
Q
Abbe’s diffraction limit:
A
- As light interacts with matter, it is not just absorbed or reflected, it is also diffracted as it passes through gaps (e.g. confocal pinhole, past organelles)
- Diffraction = scattering, loss of resolution
- Smallest distance can be resolved, based on wavelength, lens angle and numerical aperture
- …Simplifies to d = wavelength/3
- Beyond this, result is a diffuse excitation spot and a blurred image
10
Q
How can Abbe’s limit be avoided?
A
- Super-resolution techniques
- STED (i.e. principle of lasers)
- Can deplete excited state by irradiation at that energy leading to stimulated emission at different wavelength to normal fluorescence as not dropping to low vibrational level of ground electronic state
- Requires large stokes shift and broad emission bands
- Donut shaped depletion spot