Fluorescence Microscopy Flashcards
What are photons?
Energy that has no charge or no mass and travel at the speed of light and exists and EM waves
What wavelength is viable light?
750-380nm
750-red
380-blue
Define fluorescence
The emission of light by a substance that has absorbed light or other EM radiation
What was the first fluorescent substance found?
Fluorite
What are fluorophores?
Molecules able to absorb specific wavelengths of light and release energy at a different wavelength
What happens to the electrons in fluorophores?
Absorb photons which allows electrons to gain energy and enter a higher orbit and to return to their ground state they must release a photon . The photon is at a lower energy level and longer wavelength because of lost energy through molecular vibrations
What is the stokes shift?
The loss of energy from a photon within a fluorophore
What are excitation and emission wavelengths?
Light of emission wavelengths is shone onto a sample and the fluorophore generates a different emission wavelength due to stokes shift
What is a dichroic mirror?
Mirrors that can selectively reflect or block different wavelengths of light - excludes all other light except that emitted from the sample to be captured by the camera
What is a chemical fluorophore?
Compounds fused to antibodies/probs
What is a biological fluorophore?
Organsims contain chromophores to communicate in their environment e.g GFP from jellyfish
- have their genetic sequence added to a protein
What is signal bleed through?
when using more than one fluorophore and the emission wavelengths overlap and can be seen in both channels - can give rise to bleed through
What is photo-bleaching?
Prolonged exposure of fluorphores to light can damage their structure and cause photobleaching
What is prolong gold?
A slide mountant that has even engineered to polymerise intro a semi-fold matrix between a sample and a coverslip and reduce photo bleaching
