Spectral lifetime imaging

Hanley, Q. S., Arndt-Jovin, D. J. and Jovin, T. M. (2002). Spectrally resolved fluorescence lifetime imaging microscopy. Appl. Spectrosc. 56,155-66. 

Hanley, Q. S. and Ramkumar, V. (2005). An internal standardization procedure for spectrally resolved fluorescence lifetime imaging. Appl. Spectrosc. 59, 261-6. 

Pelet, S., Previte, M. J. and So, P. T. (2006). Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. J. Biomed. Opt. 11, 34017. 

M-5 (Quin-2) was the first practical fluorescent indicator for cytosolic calcium with a simple 6-methoxyquinoline as its fluorophore. Ca2+-binding increases the fluorescence intensity about six-fold (without spectral displacement, in contrast to Fura 2 see Section 10.3.3). The fluorescence lifetime of Quin-2 is highly sensitive to calcium concentration Quin-2 can thus be used as a probe in the technique of fluorescence lifetime imaging. 

A two-photon microscope with multispectral FLIM and nondescanned detection is described in [60]. An image of the back aperture of the microscope lens is projected into the input plane of a fibre. The fibre feeds the light into a polychro-mator. The spectrum is detected by a PML-16 multianode detector head, and the time-resolved images of the 16 spectral channels are recorded in an SPC-830 TCSPC module. Spectrally resolved lifetime images obtained by this instrument are shown in Fig. 5.82. 

V. Ulrich, P. Fischer, I. Riemann, K. Konig, Compact multiphoton / single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging. Scanning 26, 217-225 (2004)... 

Knemeyer, JP, Herten, DP, and Sauer, M, Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy. Analytical Chemistry 75 (2003) 2147-2153. 

Our previous approaches to detect endogenous complexes of dynamin and auxilin using co-immunoprecipitation approaches were unsuccessful, so we turned to fluorescence lifetime imaging microscopy (FLIM). While fluorescence microscopy provides two- or three-dimensional information about fiuorophore concentration, FLIM can reveal spatial differences in fluorophore population lifetimes that are independent of concentration. Besides being useful in fiuorophore identification, which transcends issues of spectral overlap, FLIM inherently observes lifetime truncations on a pixel by pixel basis that are induced by fluorescence resonance energy... 

Dumas, D. Stoltz, J. F. New tool to monitor membrane potential by FRET voltage sensitive dye (FRET-VSD) using spectral and fluorescence lifetime imaging microscopy. Clin. Hemorheol Microcirc. 2005, 33, 293-302. 

The fourth chapter by James McGuinty et al. describes the more advanced forms of time-domain FLIM. While not immediately available on commercial instruments this chapter should give the reader an idea what the current state-of-the-art is in terms of FLIM instrumentation, and perhaps what to expect on future commercial instruments. Real-time FLIM, combined FLIM-spectral imaging, hyperspectral FLIM-imaging, combined lifetime-anisotropy imaging and some of their applications are covered here. 

Instrumentally, spectral FLIM generates a spectrally resolved set of lifetimes by either introducing filters to provide spectral resolution or a spectrograph between the sample and image intensifier. The first such system was created for looking at the long lifetimes of lanthanide dyes [37]. Later, a spectral FLIM system was described for measuring from a two-dimensional (2D) area of a microscope field... 

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