BCH3021 Lecture Notes - Lecture 2: Green Fluorescent Protein, Fluorophore, Colocalization
Lecture 2 – Methods in Cell Biology: Fluorescence Microscopy
Fluorescence Microscopy – Concepts
• Fluorophore
o Compound that absorbs light of one wavelength
o Molecule that is capable of fluorescing
▪ Ground state: lower energy stable configuration (doesn’t
fluoresce)
▪ Excitation
• When light from external source hits fluorophore →
molecule absorbs light energy → energy is sufficient
reaches higher energy state: excited state
• Multiple excited states or energy levels that the
fluorophore can attain depending on wavelength and
energy of external light source
• Fluorophore is unstable at high energy configurations
→ adopts lowest energy excited state (semi-stable)
• Fluorophore rearranges from semi-stable excited state
back to the ground state → excess energy is released
and emitted as light
o Emitted light is of a longer wavelength than absorbed light
▪ Colour different
o Can be cell-permeable
o Sensitive to pH and intracellular calcium levels
o Can be coupled to proteins and other carrier molecules
o Different emission wavelengths can be used to provide information
about same sample – merging / colocalization
• Green fluorescent protein (GFP) example of a protein that has intrinsic
fluorescent properties
Fluorescence
• Absorption of light raises energy level → excited, unstable electronic state →
loses energy to lowest energy state
o Loses some energy as heat
o Can only emit light at lower energy
• Some molecules able to be excited via absorption of light energy to a higher
energy state: excited state → emission of light energy (fluorescence)
• Excitation spectrum
o Y axis: relative intensity (emitted fluorescence) vs. X axis: wavelength
(nm)
• Emission Spectrum
o Relative intensity of emitted light as a function of emission wavelength
• Process: absorption → excitation → emission (repeat)
• Intensity of emitted fluorescent light is proportional to amount of fluoropho
present
• Light source: white light requires barrier filter
Multi-Colour Fluorescence
• Fluorophores with different emission wavelengths can be used to stain
samples simultaneously
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Document Summary
Lecture 2 methods in cell biology: fluorescence microscopy. Intensity of emitted fluorescent light is proportional to amount of fluoropho present: light source: white light requires barrier filter. Multi-colour fluorescence: fluorophores with different emission wavelengths can be used to stain samples simultaneously, multiple images collected by viewing same field using different barrier filters, final picture made by overlaying separate images. Organelle/scaffolding specific fluorophores: cell permeable used to image live cells. Calcium and ph sensitive probes: fluorescence of some compounds require either calcium or specific ph, when ca is low blue fluorescence when concentration increases turns yellow. If target protein is intracellular, cells must be permeabilized to allow entry of antibody: direct immunofluorescence, primary antibody coupled to a fluorophore. Indirect immunofluorescence: primary antibody detected by a fluorescent secondary antibody, amplification, only need one secondary antibody and numerous primary antibody. Green fluorescent protein: biological fluorophore part of bioluminescence system of jellyfish, purified gfp will emit green light when exposed to blue light.
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