Frequency-Domain Fluorescence Lifetime Imaging Microscopy: A Window on the Biochemical Landscape of the Cell
Fluorescence microscopy is an established technique for determining the localization and properties of molecules in biological specimens. Obvious advantages of fluorescence are sensitivity, specificity, and spectral characteristics that depend on the environment of the probe. In addition, the low energy content of fluorescence photons in the visible part of the spectrum permits nondestructive measurements in living cells. Imaging the spatial distribution of a molecule using its fluorescence intensity has been complemented with (micro) spectroscopic techniques for studying the physical and chemical properties of the molecular environment of the fluorophore, which allow the observation of biochemical activity in cells. This has typically been achieved by exploiting the steady-state spectral characteristics of fluorescent probes that change their emission energy upon reaction with the environment. With such techniques, an image that is related to the physiological parameter of interest can be calculated from the ratio of intensities obtained at two excitation or emission wavelengths, eliminating the concentration and light path dependence of the fluorescence intensity. To quantify these images, the ratio as a function of the physiological parameter of interest has to be calibrated separately.
KeywordsFluorescence Lifetime Image Intensifier Decay Kinetic Fluorescence Lifetime Image Microscopy Homodyne Detection
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