Left: schematic diagram of HPTRF instrument, as described in text. Right: typical acquired data, showing the average of 1000 pulses (total acquisition time 0.1 s) for the IRF (light scatter from glycogen in water) and fluorescence ( anthracene in MeOH). Acquisition is triggered at by a signal from a photodiode, and excitation begins at .
Waveform precision of HPTRF. Left: the first 25 of the 1000 single-pulse waveforms acquired every 0.1 ms. Right: S/N, where S(t) is the mean signal intensity, and N(t) is the standard deviation, calculated for all 1000 waveforms. (a) anthracene in methanol. (b) rhodamine 6G in water. (c) IRF.
Linearity of waveforms. (a) Waveforms, averaged from 1000 pulses (100 ms total acquisition time), are recorded for RB (left) and Rh6G (right) with variable excitation intensity. (b) Integrated fluorescence vs excitation intensity. (c) Waveforms from (a) normalized by values in (b). All ten waveforms are plotted and overlaid, showing excellent reproducibility. (d) Standard deviations from (c).
Fluorescence waveforms acquired from anthracene by HPTRF (red, acquired from a single pulse) and TCSPC (black, 60 s acquisition time). Residuals are from single-exponential fits. Lifetime values are given as mean and standard deviation from repeated acquisitions. , where S(t) is the background-subtracted signal of a single-pulse HPTRF acquisition or a 60-s TCSPC acquisition and N(t) is the standard deviation, calculated for 1000 replicate acquisitions (HPTRF) or (TCSPC).
Lifetime analysis of standard dyes excited at (a) 355 nm (5 nM DBA black, Anth red) and (b) 532 nm ( RB blue, RB red, Rh6G black,) and analyzed using FARGOFIT. Residuals are from single exponential fits. Lifetimes are in Table I.
Fluorescence lifetimes (a) and amplitudes (b) are accurately determined by fitting waveforms to Eq. (1) over a wide range of signal intensity, which was varied by varying excitation intensity as in Fig. 3.
Analysis of dye mixtures. Left: dyes excited at 355 nm (Anth and DBA, total concentration in methanol). Right: dyes excited at 532 nm (Rh6G and RhB, total concentration in water). (a) Each waveform is the average of 1000 single-pulse waveforms, with the mole fraction of the short-lifetime dye increasing from 0 (black) to 1 (red) in 0.05 increments. (b) Lifetimes determined from independent fits (open circles) and from global fits (horizontal lines). (c) Mole fractions of the short-lifetime dye from the same fits as in (b) for independent (open circles) and global (closed circles) analysis.
Lifetimes (nanosecond) of fluorescent standards. ( of 5–8 trials.)
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