Overview
Environment Probe
Biological FLIM
3D FLIM
Diode-pumped ultrafast laser technology
Tunable visible ultrafast continuum source

Overview

Laser-induced fluorescence provides a powerful modality for biomedical applications, ranging from optical biopsy to functional imaging.Functional information may be readily derived from fluorescence lifetime due to its dependence on fluorophore radiative and non-radiative decay rates.Imaging of fluorescence lifetime therefore provides a robust means of acquiring spatially resolved information regarding the local environment of a distributed fluorophore in biological tissue and other heterogeneous or turbid media. Fluorescence lifetime probes have already been demonstrated for the measurement of e.g. [Ca²⁺], [O₂] and pH and can provide information concerning the physical properties associated with a fluorophore environment, e.g. viscosity, temperature etc.

Our basic fluorescence lifetime imaging (FLIM) set-up is illustrated in Figure 1.It is based on a commercial ultrafast Ti:Sapphire laser oscillator and a homebuilt Cr:LiSAF amplifier [1] . After frequency doubling, this delivers ~1 mJ pulses of 10 ps duration at 5 kHz repetition rate at ~415 nm. These excite a fluorescence intensity distribution in a sample, which is imaged onto a gated optical image intensifier (Kentech Instruments Ltd) that acquires whole field 2D intensity images with an effective gate width of ~ 90ps including timing jitter [2].FLIM maps are produced by acquiring a series of time-gated fluorescence intensity images at a range of time delays after excitation and, for each pixel in the field of view, fitting the assumed decay profile using the Marquardt algorithm for a nonlinear least squares fit.Depending on the samples, the autofluorescence decay is better fitted by a single or double exponential decay. The spatial variation of can then be plotted on a pseudocolor map for display.When applied to simple fluorophore distributions, this instrument can provide FLIM maps with an update time of only 3s.

Environment Probe

We have demonstrated that FLIM can be used to contrast different chemical species (i.e. different fluorophores) in a field of view and to image distributions of different fluorophores.The dependence of the fluorescence lifetime on the local environment (via its impact on the non-radiative decay rate) also permits us to image perturbations in the fluorophore environment, such as viscosity.For example, the fluorescence lifetime of the dye DASPI is a sensitive function of the viscosity of its solvent and so can be adjusted by preparing solutions in different mixtures of ethanol and glycerol. Figure 2(b) shows a FLIM map of four samples of DASPI (all at ~ 70  mM) in solvents comprising mixtures of ethanol:glycerol in the ratios shown in figure 5(a). The variation of fluorescence lifetime with viscosity is clearly apparent and demonstrates the potential of FLIM for functional imaging.This figure also illustrates the excellent temporal discrimination, which we have demonstrated to be better than ~10 ps [3].The longest fluorescence lifetime we can measure is limited only by the repetition rate of our laser system.

Biological FLIM

Our main goal in the FLIM programme is to develop novel non-invasive biomedical imaging techniques and so we have applied FLIM to autofluorescence of biological tissue.Typically we have observed that when we excite at 415 nm, we observe a complex fluorescence decay profile that we can fit to a double exponential decay.Figure 3 shows a conventional histological section of a sample of rat ear and the corresponding fluorescence intensity and lifetime maps.We obtain excellent intrinsic contrast between collagen, elastic cartilage and the veins and arteries in the tissue.These preliminary results indicate the potential of FLIM for biomedical imaging, which we hope to apply to optical biopsy for the diagnosis and monitoring of disease, as well as to provide real-time feedback of the (physical and functional) state of biological tissue during therapeutic procedures.

 

 

 

 

 

 

 

 

 

3D FLIM

For in-vivo biomedical applications it is highly desirable to perform 3D functional imaging with true whole-field parallel pixel. In a collaboration with the group of Professor Tony Wilson at Oxford University, we have realised whole-field 3D FLIM using structured illumination to achieve optical sectioning.This techniques works by utilizing the observation that it is only the zero spatial frequency components of a mage that are not attenuated with defocus in a conventional whole-field microscope.By spatially modulating the excitation and fluorescence intensity distribution, it is possible to obtain both a conventional image (with blurring from out of focus light) and a sectioned image that is similar to what would be obtained in a confocal scanning microscope.As shown in figure 4, this technique allows us to achieve both sectioned fluorescence intensity and lifetime images [4].The whole-field nature of this approach provides a relatively high frame rate, making it very attractive for FLIM of living cells and biological processes.

We now have several ongoing interdisciplinary projects to exploit FLIM for various applications.Our current programmes are principally funded by EPSRC and BBSRC and we gratefully acknowledge CASE awards from the Institute of Cancer Research and Kentech Instruments Ltd.Our current research priorities are briefly outlined below:

Diode-pumped ultrafast laser technology

To meet our aspirations to develop FLIM systems for real-world applications, it is clearly necessary to use laser technology that may become compact, portable and relatively inexpensive.We have therefore continued the tradition of the Femtosecond Optics group at Imperial College to develop an all-solid-state diode pumped oscillator amplifier system, e.g.[5].Our current system provides reliable day to day operation and is driven by just four 500 mW pump laser diodes requiring a total current of just a few amps.We hope to further develop this technology to provide a robust and low-cost alternative to existing commercial ultrafast laser technology.

Software development for improved FLIM contrast

We have recently investigated the application of a stretched exponential decay model to FLIM. We have demonstrated that, when applied to analysis of biological sample containing components associated with complex fluorescence decay profiles, it provides superior contrast and image quality, as shown in figure 5, for a minimum of computational overhead.We continue to develop this tool and to develop a robust software environment for the acquisition and analysis of time-domain FLIM data. 

Improved 3-D FLIM and multi-modal imaging

We continue to develop our 3-D FLIM instrumentation using structured illumination.We have improved our results by incorporating superior 12-bit CCD camera technology and have recently included spectral  discrimination to provide 5-D fluorescence imaging.We hope to extend this functionality and apply it to functional imaging of cells, including those labelled with green fluorescent protein and similar labels.We also intend to study tissue autofluorescence and investigate the origin of the FLIM contrast that we observe.We also intend to investigate FLIM for bio-chip assays and High Throughout Screening application.

Endoscopic FLIM

We are working to adapt FLIM to endoscopic instrumentation in order to investigate its application as a clinical tool for diagnosis and for inter-operative biopsy.We have a project targeted at imaging tissue viability and muscular degeneration and wish to use endoscopic FLIM to support our investigation of the origin of FLIM contrast in tissue autofluorescence.

References

1.Hyde, S.C.W., N.P. Barry, R. Mellish, P.M.W. French, J.R. Taylor, C.J. Vanderpoel, and A. Valster, Argon-Ion-Pumped and Diode-Pumped All-Solid-State Femtosecond Cr:LiSAF6 Regenerative Amplifiers. Optics Letters, 1995. 20(2): p. 160-162.

2.Dowling, K., S.C.W. Hyde, J.C. Dainty, P.M.W. French, and J.D. Hares, 2-D fluorescence lifetime imaging using a time-gated image intensifier. Optics Communications, 1997. 135(1-3): p. 27-31.

3.Dowling, K., M.J. Dayel, S.C.W. Hyde, J.C. Dainty, P.M.W. French, P. Vourdas, M.J. Lever, A.K.L. Dymoke-Bradshaw, J.D. Hares, and P.A. Kellett, Whole-field fluorescence lifetime imaging with picosecond resolution using ultrafast 10-kHz solid-state amplifier technology. Ieee Journal of Selected Topics in Quantum Electronics, 1998. 4(2): p. 370-375.

4.Cole, M.J., J. Siegel, S.E.D. Webb, R. Jones, K. Dowling, P.M.W. French, M.J. Lever, L.O.D. Sucharov, M.A.A. Neil, R. Juskaitis, and T. Wilson, Whole-field optically sectioned fluorescence lifetime imaging. Optics Letters, 2000. 25(18): p. 1361-1363.

5.Mellish, R., N.P. Barry, S.C.W. Hyde, R. Jones, P.M.W. French, J.R. Taylor, C.J. Van der Poel, and A. Valster, Diode-Pumped Cr-LiSAF All-Solid-State Femtosecond Oscillator and Regenerative Amplifier. Optics Letters, 1995. 20(22): p. 2312-2314.