Microspectroscopic fluorescence analysis with prism‐based imaging spectrometers: Review and current studies

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Abstract
Background: Fluorescence imaging spectroscopy is a powerful but under‐utilized tool. This article gives perspective on the use of imaging spectroscopy, and provides two examples of imaging spectroscopy done with a prism‐based system. The intent is to give insight into the power of imaging spectroscopy when used in combination with other imaging techniques. In particular, studies of intact coral photobleaching and beads designed to show energy transfer are reported. In the bead study, spectroscopic lifetime imaging was performed at each photobleaching step. Results: Spectroscopic photobleaching of the hard coral, Montastrea annularis, revealed two spectral regions. A region in the red portion of the spectrum bleached rapidly while progressively increasing fluorescence was observed over a wide portion of the spectrum. This behavior is consistent with current theories for the role of fluorescent proteins in corals. Following a photobleaching study of beads designed to exhibit energy transfer with imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM) allowed unambiguous assignment of fluorescence resonance energy transfer (FRET). The data in this experiment indicated that most of the commonly used markers of FRET would have been inconclusive. The ability of the ISFLIM to look at all regions of the spectrum, particularly the acceptor region, allowed FRET to be assigned. Conclusions: Fluorescence imaging spectroscopy is a rapidly advancing technology, uniquely suited to the flexible detection of dyes over a wide range of wavelengths. © 2006 International Society for Analytical Cytology