K. Dowling

Fluorescence lifetime imaging with picosecond resolution for biomedical applications.

We describe a novel whole-field fluorescence lifetime imaging system, based on a time-gated image intensifier and a solid-state laser oscillator-amplifier, that images lifetime differences of less than 10 ps. This system was successfully applied to discrimination between biological tissue constituents.

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

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 fluorescence 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, standard 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 potentially 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.

A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning

This article describes a wide-field time-domain fluorescence lifetime imaging (FLIM) microscope with optical sectioning. The FLIM system utilizes a wide-field time-gated optical image intensifier, with a minimum gate width of 85 ps, to achieve high temporal resolution of fluorescence decays induced by ultrashort laser pulses. Different configurations, using excitation pulses of picojoule energy at 80 MHz repetition rate and of nanojoule energy at 10 kHz, are compared. The instrument has a temporal dynamic range spanning from 100 ps to tens of μs and is shown to have a temporal discrimination better than 10 ps. When applied to laser dye samples, it has produced FLIM maps demonstrating sensitivity to variations in both chemical species and local environment, e.g., viscosity. Wide-field optical sectioning is achieved using the technique of structured illumination, which is applied to remove out-of-focus light that can result in lifetime artifacts. The sectioning strength, which may be adjusted by choosing an appropriate spatial modulation frequency, is characterized and shown to be comparable to that of a confocal microscope. Practical considerations concerned with improving the quality of sectioned fluorescence lifetime maps, including using a large bit depth camera, are discussed.

2-D fluorescence lifetime imaging using a time-gated image intensifier

Abstract We report a 2-D fluorescence lifetime imaging system based on a time-gated image intensifier and a Cr:LiSAF regenerative amplifier. We have demonstrated 185 ps temporal resolution. The deleterious effects of optical scattering are demonstrated.

Whole-field optically sectioned fluorescence lifetime imaging

We describe a novel three-dimensional fluorescence lifetime imaging microscope that exploits structured illumination to achieve whole-field sectioned fluorescence lifetime images with a spatial resolution of a few micrometers.

High resolution time-domain fluorescence lifetime imaging for biomedical applications

Abstract We report the development of a whole-field fluorescence lifetime imaging (FLIM) system with high temporal resolution based on a time-gated image intensifier. The optimized temporal gate width is < 100 ps, and changes in the environment of a fluorescent phantom, causing lifetime differences less than 10 ps, have been imaged. The versatility of this FLIM system has been demonstrated by measuring both the temporal and spectral profiles of multiple fluorescent samples in a single acquisition. Initial fluorescence lifetime images suggest that this technique can provide a means of distinguishing between different tissue constituents.

Whole-field fluorescence lifetime imaging with picosecond resolution using ultrafast 10-kHz solid-state amplifier technology

We report the development of a high temporal resolution whole-field fluorescence lifetime imaging system based on an ultrafast solid-state laser system and a time-gated image intensifier operating at up to 10 kHz. The temporal instrument response is /spl sim/110 ps and we have imaged (environmentally perturbed) differences in fluorescence lifetime as small as 20 ps. Fluorophores exhibiting single- or double-exponential fluorescence decay profiles are routinely imaged and a near real-time update time of 3 s for the fluorescence lifetime map has been demonstrated using a modest personal computer. We also present provisional fluorescence lifetime images of tissue constituents. This fluorescence lifetime imaging technology is applicable to almost any optical instrument configuration and, when coupled with existing all-solid-state diode-pumped ultrafast laser technology, may yield a potentially inexpensive instrument for in vitro and in vivo biomedical imaging.

Application of the stretched exponential function to fluorescence lifetime imaging of biological tissue

The fluorescence decay in fluorescence lifetime imaging (FLIM) is typically fitted to a multi-exponential model with discrete lifetimes. The interaction between fluorophores in heterogeneous samples (e.g. biological tissue) can, however, produce complex decay characteristics that do not correspond to such models. Although they appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, the assumption of multiple discrete components is essentially arbitrary and often erroneous. The stretched exponential function (StrEF) describes fluorescence decay profiles using a continuous lifetime distribution as has been reported for tryptophan, being one of the main fluorophores in tissue. We have demonstrated that this model represents our time-domain FLIM data better than multi-exponential discrete decay components, yielding excellent contrast in tissue discrimination without compromising the goodness of fit, and it significantly decreases the required processing time. In addition, the stretched exponential decay model can provide a direct measure of the sample heterogeneity and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

Imaging Biological Tissue Using Photorefractive Holography and Fluorescence Lifetime

This article reviews two approaches to biomedical imaging, namely photorefractive holography as a means of realising depth-resolved imaging through turbid media and fluorescence lifetime imaging as a spectroscopic imaging modality.

High-speed 3D imaging using photorefractive holography with novel low-coherence interferometers

We report high speed (~ 470 frames/s) 3-D imaging using photorefractive holography with sources of diverse temporal and spatial coherence and discuss design considerations for real-world high bit-rate imaging systems. We also propose a new real-time optical sectioning technique based on structured illumination with photorefractive holography to detect fluorescence.