Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

Background: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. Methods and Results: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simulate, display, and discuss the effects of blurring and fluorophore heterogeneity on lifetime imaging in widefield and confocal configurations. Conclusion: Sectioning improves the quality of lifetime images of samples with multiple fluorophores or spatially varying Forster resonance energy transfer. (c) 2005 International Society for Analytical Cytology.