Fluorescence Excited-State Lifetime Imaging

Time-resolved fluorescence spectroscopy is a well-established technique for studying the emission dynamics of fluorescent molecules i.e. the distribution of times between the electronic excitation of a fluorophore and the radiative decay of the electron from the excited stated producing an emitted photon. The temporal extent of this distribution is referred to as the fluorescence lifetime of the molecule. Lifetime measurements can yield information on the molecular microenvironment of a fluorescent molecule. Factors such as ionic strength, hydrophobicity, oxygen concentration, binding to macromolecules and the proximity of molecules that can deplete the excited state by resonance energy transfer can all modify the lifetime of a fluorophore. Measurements of lifetimes can therefore be used as indicators of these parameters. Furthermore, these measurements are generally absolute, being independent of the concentration of the fluorophore. This can have considerable practical advantages. For example, the intracellular concentrations of a variety of ions can be measured in vivo by fluorescence lifetime techniques (Szmacinski et al., 1994 Methods Enzymol. 240, 723). Many popular, visible wavelength calcium indicators, such as Calcium Green 1, give changes of fluorescence intensity upon binding calcium. The intensity-based calibration of these indicators is difficult and prone to errors. However, many dyes exhibit useful lifetime changes on calcium binding and therefore can be used with lifetime measurements (Lakowicz, et al., 1994 Cell Calcium 15, 7). This gives the considerable advantage that absolute measurements of concentration can be made with no elaborate calibration procedures required. Alternatively, lifetime measurements may be used to calibrate the intensity signals from these indicators when maximum sensitivity is required.

An exciting new development of the field has been the development of the technique of fluorescence lifetime imaging microscopy (Lakowicz et al., 1992 Anal. Biochem. 202: 316). In this technique lifetimes are measured at each pixel and displayed as contrast. Lifetime imaging systems have been demonstrated using wide-field (Lakowicz et al., 1992 Anal. Biochem. 202, 316-330), confocal (Sanders et al., 1995 Anal. Biochem. 227, 302-308) and multiphoton (French et al., 1997 J. Microsc. 185, 339;) imaging modes. FLIM combines the advantages of lifetime spectroscopy with fluorescence microscopy by revealing the spatial distribution of a fluorescent molecule together with information about its microenvironment. In this way an extra dimension of information is obtained. This extra dimension can be used to discriminate among multiple labels on the basis of lifetime as well as spectra. This would allow more labels to be discriminated simultaneously than by spectra alone in applications where many labels are required such as FISH, for example. There are also promising applications of lifetime imaging in the medical sciences.

We at LOCI are particularly interested in the possibilities that are opened up by multiphoton lifetime imaging of live specimens. In these applications lifetime imaging, in conjunction with spectral imaging should greatly facilitate studies using ion indicator probes and FRET studies of intermolecular distances. For example, a remarkable calcium indicator has recently been described that is a chimeric protein based on two spectrally distinct forms of fluorescent protein (cyan and yellow) and a calmodulin molecule (Miyawaki et al., 1997 Nature 388, 882). Being a naturally fluorescent protein, genetic transformants can be made so that transformed animals will express the indicator in a range of cell types determined by the promoter. The excitation wavelength is chosen to primarily excite the cyan fluorophore. On binding calcium, the calmodulin portion of the molecule changes conformation bringing the two fluorophore regions closer together allowing resonant energy transfer between the cyan and the yellow. This will cause a shift of the emitted spectrum from cyan to yellow. The development of this engineered protein (known as Cameleon) is a remarkable development as it circumvents all the problems associated with loading probes into cells since stable transgenic lines can be used which all express Cameleon. However, one of the problems with Cameleon is that, although ratiometric methods can be used, the signal change on binding calcium is quite small making this indicator less sensitive than other indicators such as Calcium Green. Lifetime measurements are a sensitive indicator of FRET (Godella et al., 1995 J. Cell Biol. 129, 1543) and in combination with spectral measurements, should provide a more sensitive indication of calcium levels.