Part 2A: Background Flashcards
Describe fluorescence imaging
Detection of light emitted from a sample upon irradiation
Advantages of fluorescence imaging
- Better contrast than offered by reflection or transmission alone
- Can achieve different localisation, including with several emissive species
Outline the two key types of fluorescence microscopy
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
Pixel, Voxel
- Pixel: Spot in xy plane
- Voxel: Reconstituted 3D point
Confocal microscopy: 2 advantages and 2 disadvantages
+ Very high signal:noise ratio
+ High resolution
- Limited tissue penetration (<1mm)
- Abbe’s diffraction limit
Give 5 properties of useful fluorophores
- 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)
How do fluorophores operate?
- 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)
Give 4 essential properties of luminophores:
- 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
Typical commercially available fluorophores, give an example
- 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
How do charge transfer fluorophores operate?
- Charge transfer from electron rich to electron poor
- Photo-induced charge transfer leads to a charge-separated excited state
What are fluorophores typically responsive to?
- pH responsive e.g. fluorescein
- Potential responsive
- Analyte responsive
pH responsive agents:
- 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
Potential responsive agents:
- 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
Analyte responsive agents:
- 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
Ratiometric sensing:
- Different wavelength with and without analyte
- Using ratios to derive concentration of analyte
- Utilises calibration curve
