Fast-acquisition multispectral FLIM by parallel TCSPC

Currently used TCSPC FLIM systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. The systems are, however, restricted to count rates on the order of a few MHz. In the majority of applications, such as FRET or tissue autofluorescence, the photostability of the samples limits the count rate to much lower values. The limited counting capability of the hardware is therefore no problem. However, if FLIM is used for samples containing highly photostable fluorophores at high concentrations the available count rates can exceed the counting capability of a single TCSPC channel. In this paper we describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 50•106 s-1. By using a polychromator for spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live plants.