Iain Johnson
Molecular Probes, Inc.
4849 Pitchford Avenue
Eugene, OR 97402-9144

1. Fluorescence The Three Prerequisites: - light source, fluorophore and photon detector. Electronic transitions: - the Jablonski diagram - absorption (extinction coefficient) - excited states - emission (fluorescence quantumyield) - alternate deactivation pathways. Spectra: - absorption spectra - fluorescence excitation and emission spectra - the fluorescence Stokes shift - spectral bandwidths.

2. Fluorophores and Fluorescent Probes Characteristics of fluorophores: - spectra - extinction coefficients - fluorescence quantum yields. Photobleaching. Environmental sensitivity of fluorophores: - quenching - solvent shifts - pH effects. Fluorescent tracers: fluorescent microsphere technology.

3. Fluorescence Instrumentation Spectrofluorometers - comparison with spectrophotometers - capabilities and performance of contemporary instruments. Microtiter plate readers/scanners. Flow cytometers. Microscopes.

4. Analytical Fluorometry Relationship between fluorescence and analyte concentration: - nonlinearities (inner filter effects) - sensitivity. Background: - autofluorescence - scattered excitation light. Standardization: -reporting fluorescence intensities. Resolution of multicomponent mixtures: -synchronous scanning.

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