Abstract
We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back‐aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16‐anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time‐correlated single photon counting (TCSPC) process. The recording process builds up a four‐dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near‐ideal counting efficiency and is capable of resolving double‐exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue. Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.