Fluorescence Lifetime Imaging for Diagnostic and Therapeutic Intravital Microscopy

Abstract

Intravital imaging is now widely performed using wide-field microscopy, endoscopy, and state-of-the-art multiphoton microscopy for research and clinical assessment applications. Fluorescence lifetime imaging is increasingly being used as a complementary technology to greatly enhance the specificity and sensitivity in the analysis of the various fluorophores present within an intravital image. The fluorescence lifetime of a fluorophore. The fluorescence lifetime distribution for a fluorophore is an intrinsic property, arising from the emission of photons of light in the decaying to its original energy state after its molecules are excited by a specific wavelength of light and remain in an excited state for a range of times. This behavior for individual autofluorescent fluorophores, dyes, drugs, fluorescent proteins and antibodies is most frequently summarized in terms of their average fluorescence lifetime. Fluorescence lifetime differences are then used to identify and discriminate between molecules in various applications, including the assessment of drug distribution and metabolism, and in quantifying cell responses for toxicology. Fluorescence lifetime imaging microscopy (FLIM) and tomography involves the spatial representation of the fluorescent lifetimes of all molecules within image collected over a specified time period and resolution. Autofluorescence lifetime differences between normal and cancerous tissues have been used to define surgical margins during intraoperative surgery. Recent advances have enabled the rapid and robust collection of fluorescence lifetime information from tissues with high-resolution at video-rate speeds using endoscopic probes. Fluorescence lifetime imaging, combined with multi-spectral and anisotropic analysis, yields detailed redox state data from within a cell, arising from its metabolic state and enables intravital analysis of the transport and metabolism of fluorescent probes in cells. Intravital fluorescence lifetime imaging is becoming an indispensable diagnostic approach with broad therapeutic and clinical applications.