Journal of Microscopy, Vol. 203, Pt 3, September 2001, pp. 246±257. Received 18 September 2000; accepted 21 November 2000 Time-domain whole-field fluorescence lifetime imaging with optical sectioning M. J. COLE*, J. SIEGEL*, S. E. D. WEBB*, R. JONES*, K. DOWLING*, M. J. DAYEL*, D. PARSONS-KARAVASSILIS*, P. M. W. FRENCH*, M. J. LEVER², L. O. D. SUCHAROV³, M. A. A. NEIL³, R. JUS Ï KAITIS³ & T. WILSON³ *Femtosecond Optics Group, Department of Physics, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BW, U.K. ²Department of Biological and Medical Systems, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2BY, U.K. ³Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, U.K. Key words. 3D microscopy, diode-pumped laser, fluorescence lifetime imaging, optical sectioning, whole-field imaging. Summary A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode- locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluor- escence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, stan- dard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using poten- tially compact, portable and low cost all-solid-state diode- pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique. 1. Introduction Ideally, diagnostic instrumentation for biomedical appli- cations should non-invasively provide high-resolution images with three-dimensional (3D) imaging capability and the possibility of obtaining functional information. Any radiation source should be safe for repeated exposure and the apparatus should preferably be compact, portable and low-cost. Optical imaging can potentially fulfil all of these requirements. To this end we are developing fluorescence lifetime imaging instrumentation for microscopy and for in vivo external and endoscopic application. Quantitative functional imaging is regularly realized using fluorescence, exploiting the photo-physical properties of both exogenous and endogenous fluorophores to map the chemical and/or physical properties of a sample. Unfortu- nately, excitation intensity fluctuations, sample heteroge- neity, absorption, strong scattering and background fluorescence often degrade absolute quantitative measure- ments of fluorescence intensity. To overcome these prob- lems, wavelength ratiometric techniques are often employed (Tsien & Poenie, 1986), for which only relative intensity measurements are required. Although this is a powerful approach, only a limited number of suitable wavelength- ratiometric fluorescence probes have been identified. These probes often require ultraviolet (UV) excitation, which limits the penetration depth in tissue and can lead to unwanted autofluorescence of the specimen, thus affecting the signal- to-noise ratio. Additionally, this method is sensitive to chromatic aberration and requires a complex calibration procedure. These drawbacks may be overcome by measur- ing fluorescence lifetime. Fluorescence lifetime measure- ments are also derived from relative intensity measurements Correspondence to: P. M. W. French. Tel.: 144 (0)20 7594 7706; fax: 144 (0)20 7594 7782; e-mail: paul.french@ic.ac.uk 246 q 2001 The Royal Microscopical Society