Sensing cellular function and molecular activity in vivo using fluorescence lifetime imaging microscopy (FLIM)

In contrast to intensity-based fluorescence microscopy, fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited-state lifetime. This technique is sensitive to the fluorophore's local environment (temperature, ion concentration, dissolved gas concentration, and molecular associations), while being independent of factors impacting fluorescence intensity (fluorophore concentration, photobleaching, scattering, and absorption). We present design features of a novel UV-visible-NIR wide-field time-domain FLIM system with optical sectioning (10 μm), high temporal discrimination (50 ps), and large temporal dynamic range (750 ps - 1 μs), and apply the system to probe cellular metabolic function and detect molecular activity in vivo.