Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning.

We report a novel whole-field three-dimensional fluorescence lifetime imaging microscope that incoporates multispectral imaging to provide five-dimensional (5-D) fluorescence microscopy. This instrument, which can acquire a 5-D data set in less than a minute, is based on potentially compact and inexpensive diode-pumped solid-state laser technology. We demonstrate that spectral discrimination as well as optical sectioning minimize artifacts in lifetime determination and illustrate how spectral discrimination improves the lifetime contrast of biological tissue.