title: A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode.
abstract: We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon 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 the photon 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 corresponding TCSPC 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 200-250 μm. The data acquisition software builds up the distribution of the photons over their spatial 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 to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.
authors: Becker & Hickl GmbH, Nahmitzer Damm 30, 12277 Berlin, Germany.
authors: Wolfgang
authors: W
authors: Becker
authors: Department of Physics, King's College London, Strand, London WC2R 2LS, United Kingdom.
authors: Liisa M
authors: LM
authors: Hirvonen
authors: Photek Ltd., 26 Castleham Rd., Saint Leonards-on-Sea TN38 9NS, United Kingdom.
authors: James
authors: J
authors: Milnes
authors: Photek Ltd., 26 Castleham Rd., Saint Leonards-on-Sea TN38 9NS, United Kingdom.
authors: Thomas
authors: T
authors: Conneely
authors: Institut für Kernphysik, Max-von-Laue-Str. 1, 60438 Frankfurt, Germany.
authors: Ottmar
authors: O
authors: Jagutzki
authors: Becker & Hickl GmbH, Nahmitzer Damm 30, 12277 Berlin, Germany.
authors: Holger
authors: H
authors: Netz
authors: Becker & Hickl GmbH, Nahmitzer Damm 30, 12277 Berlin, Germany.
authors: Stefan
authors: S
authors: Smietana
authors: Department of Physics, King's College London, Strand, London WC2R 2LS, United Kingdom.
authors: Klaus
authors: K
authors: Suhling