Application Note eight-channel-flim-apn01.doc Oct. 2009 1 An 8-Channel Parallel Multispectral TCSPC FLIM System Wolfgang Becker Abstract. 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10 6 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. Count Rates in TCSPC FLIM Experiments TCSPC FLIM [3, 6] delivers single- and multi-wavelength fluorescence lifetime images at an accuracy essentially limited by the photon statistics [1, 11], and a time resolution limited by the transit-time-spread of the detector [4, 5, 12]. This is a clear advantage in the typical FLIM applications, such as FRET measurements, investigation of membrane proteins, or autofluorescence imaging. All these applications have in common that the fluorophore concentration is low. Moreover, the excitation power has to be kept low to avoid cell damage and lifetime changes by photobleaching. The count rates available from the samples are therefore on the order of only 10 4 s -1 to a few 10 5 s -1 . This is one to two orders of magnitude lower than the counting capability of a single TCSPC FLIM device [5]. Consequently, the acquisition time required to achieve a given lifetime accuracy is exclusively given by the photostability of the sample, not by the counting capability of the TCSPC module. Technical efforts to increase the counting capability of the TCSPC FLIM electronics therefore do not result in shorter acquisition times in these applications. There are, however, certain classes of FLIM experiments that can be run at very high count rate. One of these is imaging of chlorophyll in plant tissue. Chlorophyll is highly photostable and emits strong fluorescence around 700 nm. The fluorescence lifetime of chlorophyll in live plants changes with the time of exposure [9]. Recording these changes requires fast FLIM [10]. Another potential application is, surprisingly, autofluorescence. Autofluorescence signals are usually weak, but exceptions do exist. Fig. 1 shows autofluorescence FLIM images of live yeast cells. The bright cells (probably apoptotic ones) are about 50 times brighter than the dim ones (visible only in Fig. 1, right). Fig. 1: FLIM images of yeast cells. Left: Intensity scale 0 to 3000 counts per pixel. Right: Intensity scale 0 to 300 counts per pixel. The bright cells are about 50 times brighter than the dim ones. Becker & Hickl DCS-120 confocal scanning FLIM system, excitation at 407 nm.