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Bigger and Better Photons: The Road to Great FLIM Results

This brochure is an attempt to help existing and future users of the bh FLIM technique obtain the best possible results from their FLIM experiments. The first part of the brochure explains the principle of TCSPC FLIM, and gives an impression of the photon distributions recorded. It shows that the signal-to-noise ratio of the measured lifetimes depends, in first order, on the number of photons recorded. The following sections focus on optimising the photon number without increasing the photostress imposed to the sample. We discuss the influence of excitation power, acquisition time, collection efficiency, numerical aperture, focusing precision, alignment accuracy, and detector efficiency. The next section concentrates on photon efficiency. It considers TCSPC timing parameters, counting background, number of pixels, the influence of the instrument-response function, and the challenges of multi-exponential decay functions. The last section is dedicated to data analysis. All conclusions made in this brochure are demonstrated on real measurement data recorded under realistic conditions.

Keywords: TCSPC, FLIM, Signal-to-Noise Ratio, IRF, Acquisition Time, Time Resolution, FLIM Data Analysis

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