A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

We report on the implementation of a wide-field time-correlated singlephoton counting(TCSPC) method forfluorescence lifetimeimaging (FLIM). It is based on a 40 mm diameter crossed delay lineanodedetector, where the readout is performed by three standardTCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. Thephoton arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of thephoton event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the “start” input of the correspondingTCSPC board, and the other end is delayed by 40 ns and fed into the “stop” input. The time between start and stop is directly converted into position, with a resolution of 200–250μm. Thedata acquisitionsoftware builds up the distribution of thephotons over theirspatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system tofluorescence lifetimeimaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to otherfluorescence microscopy methods.

Source: Review of Scientific Instruments - September 23, 2016 Category: Physics Authors: Wolfgang Becker, Liisa M. Hirvonen, James Milnes, Thomas Conneely, Ottmar Jagutzki, Holger Netz, Stefan Smietana and Klaus Suhling Source Type: research