M.J. Cole

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.

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.

Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning

We report a novel whole-field three-dimensional fluorescence lifetime imaging microscope that incoporates multispectral imaging to provide five-dimensional (5-D) fluorescence microscopy. This instrument, which can acquire a 5-D data set in less than a minute, is based on potentially compact and inexpensive diode-pumped solid-state laser technology. We demonstrate that spectral discrimination as well as optical sectioning minimize artifacts in lifetime determination and illustrate how spectral discrimination improves the lifetime contrast of biological tissue.

Wide-field fluorescence lifetime imaging with optical sectioning and spectral resolution applied to biological samples

Abstract. Wide-field fluorescence lifetime imaging with spectral resolution and optical sectioning has been performed to achieve five-dimensional fluorescence microscopy. Spectral filtering has been shown to have the potential to provide functional information about biological tissue by simultaneously measuring the spectral/lifetime signature of the sample. The potential to use multispectral imaging to separate cellular components spatially by their different emission wavelengths has also been demonstrated thus reducing artefacts in the calculated lifetime maps. The instrument is based on diode-pumped solid-state laser technology and an ultrafast gated optical image intensifier. Also reported is the use of a picosecond blue laser diode as the excitation source to produce a fluorescence lifetime microscope with a footprint of less than 0.25m2.

Diode-pumped all-solid-state ultrafast Cr:LiSGAF laser oscillator–amplifier system applied to laser ablation

Abstract We describe a multi-kHz repetition rate ultrafast all-solid-state diode-pumped oscillator–regenerative amplifier laser system that we have applied to ultrafast laser ablation. The oscillator was a Kerr lens mode-locked Cr:LiSGAF laser and the regenerative amplifier utilised Cr:LiSGAF in a simple three-mirror cavity. The whole laser system, which was pumped by less than 2 W total pump power from four commercially available 670 nm diodes, produced ∼1 μJ pulses at up to 10 kHz repetition rate, tunable in the near infrared. A simple double pass two grating compressor was used to adjust the pulse duration from 15 ps to 150 fs. Using this laser system we performed ablation of stainless steel and fused silica and demonstrated the characteristic pulse duration dependence of the ablation threshold for dielectrics.

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.

Fluorescence lifetime imaging for biomedicine using all-solid state ultrafast laser technology

We present potential biomedical applications for a diode- pumped ultrafast Cr:LiSGAF oscillator-amplifier system. A whole-field fluorescence lifetime imaging system has been demonstrated for the first time using such a laser system. Fluorescence lifetime imaging of unstained biological tissue in vitro using this instrument has shown contrast between different tissue constituents. Initial results of applying this laser system to the ablation of glass are also presented.

Five-dimensional fluorescence microscopy

We report a whole-field fluorescence imaging microscope that combines 3-D spatial resolution by optical sectioning, using structured illumination, with fluorescence lifetime imaging and spectrally-resolved imaging. We show the potential of this technique in the elimination of common artefacts in fluorescence lifetime imaging and apply it to study the dependence of the lifetime on the emission wavelength in biological tissue.