Fluorescence Lifetime Imaging (FLIM)
Fluorescence lifetime may be measured in the frequency or time domains and implemented in wide-field or laser scanning microscopes. In the Photonics Group we tend to work with time-domain techniques, namely wide-field time-gated imaging using a gated optical intensifier (GOI) and time-correlated single photon counting (TCSPC) which is single point measurement technique that can be applied to laser scanning confocal or multiphoton microscopy. Figure (a) shows a schematic of a wide-field time domain FLIM system1 in which the sample is excited by a series of ultrashort optical pulses from an ultrafast laser and the resulting fluorescence decay is sampled by recording the whole-field 2-D fluorescence intensity distribution at various delays (t1, t2, t3, ..) after excitation, using a gated optical intensifier with an ultrashort (~100’s ps) gate width, which acts like an ultrafast shutter. For each pixel in the field of view, the fluorescence decay constant, τ, is calculated and the results are plotted in a fluorescence lifetime map. For the simple case depicted in the figure, there are two cuvettes of different fluorophores in the field of view and these are contrasted by their different lifetimes.
In the frequency domain wide-field FLIM can be implemented with sinusoidally modulated excitation light and the fluorescence lifetime can be determined by measuring the phase of the resulting fluorescence emission, which will be modulated at the same frequency. If the fluorophores exhibit a single exponential decay constant, τ, then the measured phase shift will simply be given by tan ϕ = ωτ. The phase measurement can be made across the field of view by applying a sinusoidal modulation to the gain of an image intensifier or to a CMOS camera2. It is also possible to implement frequency domain FLIM using pulsed excitation, e.g. from a mode-locked laser, with sinusoidally modulated detection.
There are both time domain and frequency domain approaches that can be implemented in laser scanning microscopes, such as confocal and multiphoton microscopes. The first FLIM experiments with biological samples were demonstrated using time-correlated single photon counting (TCSPC)3, which involves exciting the sample at sufficiently low excitation power such that the fluorescence photons can be detected one at a time and their arrival times recorded in a histogram that provides the fluorescence decay profile. This is illustrated in figure (b). Subsequently the development of convenient and relatively low-cost TCSPC electronics, e.g.4,5, for laser scanning confocal and two-photon fluorescence scanning microscopes has stimulated the widespread uptake of FLIM. Today laser scanning FLIM microscopy provides the most efficient detection of fluorescence signals although the sequential pixel acquisition leads to relatively long FLIM acquisition times. Wide-field FLIM provides faster imaging and rapid optically sectioned FLIM can be realised by combining wide-field FLIM detection with a spinning disc quasi-confocal microscope6. Figure 5(a) shows an optically sectioned fluorescence lifetime image of live cells acquired in just 5 s and figure 5(b) shows an optically sectioned fluorescence lifetime image of live cells acquired at 5 frames/second. This rapid FLIM technology forms the basis of the high content analysis platform that we are developing to assay protein interactions.
The figures below show some examples of experimental fluorescence lifetime images labelled biological samples and of tissue autofluorescence.
1 Dowling, K. et al. Opt. Commun. 135 (1997) 27
2 Esposito, A., Oggier, T., Gerritsen, H. C., Lustenberger, F. and Wouters, F. S., Optics Express, 13 (2005) 9812
3 Bugiel, I., König, K. and Wabnitz, H., Lasers in the Life Sciences, 3 (1989) 47
4 Becker, W., Bergmann, A., Hink, M. A., Konig, K., Benndorf, K. and Biskup, C., Microscopy Research and Technique, 63 (2004) 58
5 Zhang, Y. L., Soper, S. A., Middendorf, L. R., Wurm, J. A., Erdmann, R. and Wahl, M., Applied Spectroscopy, 53 (1999) 497
6 Grant, D. M., McGinty, J., McGhee, E. J., Bunney, T. D., Owen, D. M., Talbot, C. B., Zhang, W., Kumar, S., Munro, I., Lanigan, P. M. P., Kennedy, G. T., Dunsby, C., Magee, A. I., Courtney, P., Katan, M., Neil, M. A. A. and French, P. M. W., Optics Express, 15 (2007) 15656