Fluorescence excited-state lifetime imaging
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 about 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; Wang et al., 1992. Crit. Rev. Anal. Chem. 23: 369; Gadella et al., 1993. J. Cell Biol. 129, 1543). 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), confocal (Sanders et al., 1995 Anal. Biochem. 227: 302), 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. This extra dimension of information 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. There are also promising applications of lifetime imaging in the medical sciences. For example, tumors have been detected in mice sensitized with a hematoporphyrin derivative by lifetime imaging (Cubeddu et al., 1997 Photochem Photobiol 66(2):229).
We are particularly interested in the possibilities that are offered 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 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 expressing Cameleon can be used. However, one of the problems with Cameleon is 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.
Techniques for lifetime imaging
Fluorescent lifetimes can be measured either in the frequency domain or in the temporal domain. Three general strategies have been used to measure fluorescence lifetimes:
In this scheme a high-frequency, modulated light source is used for fluorophore excitation. By the use of a gain-modulated detector, the phase shift and amplitude demodulation of the fluorescence signal is determined. From these data the fluorescent lifetime of the probe can be calculated. This scheme is robust and has been extensively used (Wang et al., 1992). However for our purposes it suffers from several drawbacks: the detector is only working at 50% of its maximum efficiency because it is gain modulated, several data sets taken at different excitation modulation frequencies have to be taken in order to separate two or more lifetime components, and this scheme does not work well with photon counting techniques which we favor.
Time-domain lifetime imaging with gated detectors
In this scheme a gated micro-channel plate image intensifier is used in conjunction with a CCD imaging camera (e.g. Straub and Hell, 1998. Applied Physics Letters 73:1769). Spectral information is obtained by gating the image intensifier on for a narrow time-window at progressively later intervals after the excitation pulse in a succession of data frame captures. This scheme is probably the simplest way of implementing a life-time imaging system. However, it suffers from two major drawbacks for our application. The method has very poor photon utilization as only one temporal interval is detected at a time. If there are 32 intervals for example, 31/32 of the signal is not utilized and 32 separate frames have to be captured. The second reason this scheme is not appropriate for a multiphoton imaging application is that an imaging (i.e. area) detector is used. This means that the deep sectioning advantage of multiphoton imaging are not fully realized because scattered fluorescence emission photons will give rise to background noise rather than contributing to the signal as can be done with a point-scanning multiphoton system.
Time-domain lifetime imaging with photon counting
For working at low-light levels, photon-counting detectors have considerable advantages in that they can virtually eliminate noise contributions from electronic amplifiers or electron multiplier noise in a photomultiplier. Also, photon-counting systems provide quantized pulses for every detected photon, allowing the lifetimes to be measured directly using electronic circuitry. Because of the very high speeds necessary to obtain sub-nanosecond temporal resolution., time-to-voltage converters are usually used to measure the interval between the fluorophore excitation pulse and the time of detection of the emitted fluorescent photon. Such schemes have been successfully used in practical photon-counting lifetime detectors (Kelly et al., 1997. Rev. Sci. Instrum 66(6):2279). These schemes are attractive because of their efficient utilization of detected photons. However they suffer from dynamic range problems that arise out of limited counting speeds. Typically, a time-to-voltage converter together with an associated analogue (voltage) to digital converter would have a maximum counting rate of around 1Mhz. Also, with this scheme, only one photon can be measured in the interval between laser pulses. These limitations restrict the use of this technique to low light levels when fairly long exposure times are needed in order to obtain sufficient counts for accurate representation of the decay curve. The comparatively large dead-time of this technique can have more insidious consequences. Immediately after the laser pulse, photons will be emitted at the highest rate and therefore more will be preferentially lost at this time because of the dead-time of the detector. This effect can distort the shape of the decay profile.