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Recoding and analyzing fluorescence decays
- Please also refer to
- “The bh TCSPC handbook” published by Becker & Hickl, e.g. p. 61-65, 259-272
- “Principles of Fluorescence Spectroscopy” by Joseph R. Lakowicz, chapter 1, 2.1, 4, 8, 10, 11, 13 & 14
- The “TCSPC technote” from Picoquant
- Time-resolved fluorescence spectrometer, e.g., FluoroMax4 equipped with the IBH time-correlated single photon counting module and the pulsed light source NanoLED
- Fluorescence cuvette (L-shaped excitation & emission configuration)
- Buffer for sample dilution
- Samples: Single donor labeled (“Donly”, D0) and double donor-acceptor (“DA”) labeled
- Ludox (colloidal silica solution)
Time-resolved fluorescence intensities are measured via time-correlated single photon counting (TCSPC) using a pulsed excitation light source and a single-photon sensitive detector. The arrival time of the emitted fluorescence photon is recorded with picosecond accuracy with respect to the incoming excitation light.
Repeating the excitation-emission process many times will give a decay profile. Pulsed lasers or LEDs can be used as a source of excitation. The excitation light is passed through a polarizer before it excites the sample in the cuvette. Part of the light passes through the sample, the fluorescence light is registered by the counting electronics as "sync" signal. The light emitted by the sample molecule is passed first through a polarizer and then through a monochromator to select a specific wavelength (~range). The light is then detected.
The characteristic of a complete TCSPC system that summarizes its overall timing precision is its Instrument Response Function (IRF or Prompt). The basic idea is that if the system is ideal, i.e., has an infinitely sharp excitation pulse and infinitely accurate detectors and electronics, it should have an infinitely narrow IRF. Any deviation from this ideal results in a broadening of the IRF.
For comparability, the full-width of half-maximum (FWHM) is calculated. For the IRF measurement, a diluted solution of LUDOX (colloidal silica) is used. It should be diluted such that its count rate is comparable to the count rate of the samples and must be measured under identical conditions as the sample (i.e. polarization, slit width). The only difference is its detection wavelength. As LUDOX is not fluorescent, and only scatters the light, it must be recorded at the excitation wavelength.