Equipment available: Horiba FluoroMax-4 Spectrofluorometer
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £45/day
Fluorescence occurs when light is emitted from an excited state. Two processes are involved: absorption and subsequent emission. Fluorophores occur naturally e.g. GFP, tryptophan, the Y-base of t-RNA, NADH, and chlorophyll, and there are a wide range of synthetic fluorescent probes e.g. fluorescein, that can be coupled to the system being studied.
Red and blue shifts of the fluorophore emission spectra, e.g. tryptophan, reflect the changing environment of the probe and can be used to follow binding events.
Quenching. Depopulation of the excited state can also occur by radiationless processes that will decrease the observed fluorescence intensity and the lifetime. Common quenchers include oxygen, I- and Cs+ and can give information on the dynamics or accessibility of the fluorophore.
Polarisation or anisotropy. If the chromophore is excited with plane polarized light and the fluorescence is observed through analysing polarizers, then it is found that the fluorescence is also polarised. . If the molecules move (e.g. by Brownian motion) during the lifetime of the fluorescence, the polarisation will be reduced by an amount that depends on the degree of motion (slow motion, little depolarisation; fast motion, strong depolarisation). Measurement of fluorescence depolarization is a powerful tool for measuring molecular motion and ligand binding. Ligand binding can be followed if the fluorophore motion is significantly slowed on binding resulting in the depolarisation going from strong to little. The depolarisation will thus give a measure of binding but this depends on the fluorophore being significantly smaller than its binding partner e.g. peptide binding to protein.
Fluorescence resonance energy transfer (FRET). Fluorescence can be lost by the transfer of excitation energy to other fluorophores (acceptors). The most common type is fluorescence resonance energy transfer (FRET) from the excited singlet state of a donor to the excited singlet state of an acceptor. In this transfer of energy, the donor returns from the excited state to the ground state and the acceptor is simultaneously excited from its ground to its excited state. The energy separations in each case must match; i.e. be in resonance. If the acceptor fluoresces, the original excitation energy can reappear as acceptor emission. When the acceptor molecule is non-fluorescent, the transferred energy is dissipated by non-radiative processes. This can be used as a molecular ruler or to follow conformational changes in proteins.
The FluoroMax is a general purpose fluorimeter. Fluorescence, phosphorescence and bio- and chemi-luminescence measurements can be made on this instrument. Sample temperature can be controlled using the attached water bath.
Requirements for sample
The sensitivity of fluorescence means that fluorophore concentrations can be usually nanomolar or lower. The facility has a range of cuvettes for general use but we would recommend frequent users buy their own to ensure that they are available and clean for their experiments.
Cuvettes for fluorescence measurements: 1ml (can be stirred in instrument). 0.5ml and 50ul. The instrument takes a 1cm square cuvettes with the beam height centre at 15mm from the bottom of the cuvette holder.
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