Spectral FLIM

Spectral FLIM involves measuring the apparent lifetimes in a preparation at many wavelengths with the assistance of a spectrograph or a series of filters (see also Chapter 4, Figs. 4.7 and 4.8 depicting hyperspectral FLIM in the time domain). The goal of the measurement is similar to that of the multifrequency approach ... 

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... 

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. 

While VFPs have boosted the applications of FRET-FLIM, chemical FRET probes should not be dismissed. The advantage of chemical probes is that they are much smaller in size and that they often have much better spectral readout than VFP probes. In Chapter 6, Amanda Cobos Correa and Carsten Schultz highlight the various small molecule-based FRET probes and their use in bioimaging. 

FIGURE 3.25. Spectral reflectance in colored and bleached states for an asymmetric electrochromic device with hydrated dielectric thin-flim ion conductors the design is sketched in the inset [3.94]. 

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. 

K. Konig, I. Riemann, G. Ehrlich, V. Ulrich, P. Fischer, Multiphoton FLIM and spectral imaging of cells and tissue, Proc. SPIE 5323, 240-251 (2004)... 

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... 

Analogous to this development is the combination of spectrally measurements with FLIM (sFLIM, [58,59]), resulting in spectrally resolved lifetimes. In practice, this can be done relatively easy, by inserting a spectrograph or dispersing element between the object and the detector. 

FLIM is not the only technique for observing FRET. If both donor and acceptor chromophores are fluorophores, FRET can also be estimated with steady state techniques, including acceptor photobleaching [102,103], spectral imaging [61,104] and ratio-imaging (or filter-FRET) [105,106] techniques. Recently all possible modes of FRET-microscopy (also including yet unexplored FRET-imaging modes) have been reviewed by Erijman and Jovin [107]. 

With each method of FRET estimation there are advantages and disadvantages. Here, specifically the three most widely used other FRET-imaging techniques besides FRET-FLIM i) acceptor-bleaching, ii) filter-FRET and iii) spectral imaging are briefly discussed in relation to FLIM. Also elsewhere, reviews and experimental studies have appeared comparing FLIM with other FRET-microscopy techniques [61,63,108]. 

Since GFPs display a relatively constant lifetime in cells (unless they are involved in FRET), FLIM has been applied for colocalization studies of spectrally similar GFPs by exploiting the hfetime-difference between these GFP-variants [144]. By assiuning constant hfetimes for each GFP-variant, global analysis... 

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