Fluorescence microscopy Flashcards
1
Q
How does the energy level of photons change in relation to wavelength of light?
A
- shorter wavelength/larger amplitude = higher energy photons
- longer wavelength/smaller amplitude = lower energy photons
2
Q
Define fluorescence
A
- emission of light by a substance that has absorbed light or other electromagnetic radiation
3
Q
What is a fluorophore and how do they emit fluorescence?
A
- specialised molecules able to absorb specific wavelengths of light and release this energy at a different wavelength
- absorption of these photons causes fluorophore atoms to gain energy and enter higher energy state or orbit
- to return to ground state must release energy as another proton
- net result is fluorescence, where photon emitted exists at lower energy level and longer wavelength than that which excited fluorophore
4
Q
What is Stoke’s shift?
A
- loss of energy from a photon within a fluorophore
5
Q
What are dichroic mirrors?
A
- specialised mirrors able to selectively reflect/block passage of different
wavelengths of light - allow quick switching between filters when using multiple fluorophores
6
Q
What is a chemical fluorophore?
A
- fused to purified proteins, eg. Abs, FISH probes and DNA oligonucleotide primers
- eg. DAPI
7
Q
What are biological fluorophores?
A
- chromophores
- eg. GFP
- can be added to any protein in genome and visualised in live cells
8
Q
Why is signal bleed through an issue?
A
- if using multiple fluorophores essential they have distinct excitation values
- can cause false signals, making interpretation and analysis difficult
9
Q
What is photo-bleaching?
A
- reduction in fluorescence due to prolonged exposure to high intensity light, which damages their structure
10
Q
How can photo-bleaching be reduced?
A
- specialised mountants = compounds that polymerise into semi-solid matrix between sample and coverslip
- eg. Prolong Gold
11
Q
Why is it important for exposure/gain conditions to be constant between slides?
A
- to account for background fluorescence
