D. Parsons-Karavassilis

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.

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.

High frame-rate, 3-D photorefractive holography through turbid media with arbitrary sources, and photorefractive structured illumination

In this paper, we briefly review our work on low-coherence photorefractive holography and report on the current state of the art. We present what is, to the best of our knowledge, the fastest-ever three-dimensional (3-D) imaging system and present results obtained with imaging at 470 frames/s (fps). We demonstrate the versatility of photorefractive holography using various sources, including LEDs, high-power diode arrays, and a novel, all solid-state broad-band laser. We present preliminary results obtained by combining the technique of structured illumination with photorefractive holography for the first time. We demonstrate that this novel holographic optical sectioning technique may be implemented for both reflection and fluorescence imaging.

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.

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

The paper reports recent progress in developing high speed 3D imaging systems based on low coherence photorefractive holography with high-speed depth-sectioned imaging at 476 frames per second. It is demonstrated that photorefractive holography can utilize a wide variety of sources of differing spatial and temporal coherence, including a novel all-solid-state broadband laser. Also presented is a novel real-time optical sectioning technique based on structured illumination combined with photorefractive holography that provides real-time optical sectioning when imaging with reflected light or with fluorescence.

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.

Low-coherence photorefractive holography for high-speed 3D imaging including through scattering media

Low coherence photorefractive holography is a wide-field technique for 3-D imaging that offers a unique mechanism to discriminate against a background of diffuse light. This provides a wide-field method to image through scattering materials that we have demonstrated may be implemented at frame rates as high as ~ 470/second. We present our recently developed low coherence photorefractive microscope and demonstrate how it may be realized using a spatially coherent broadband c.w. diode-pumped solid-state laser. This can provide real-time sectioned images of moving 3-D objects using only a simple uncooled 8-bit CCD camera. We also demonstrate a photorefractive 3-D imaging technique that exploits structured illumination and photorefractive holography to achieve a real-time wide-field sectioning microscope that may be applied to fluorescence, as well as reflected light. We also discuss issues for improving the sensitivity and spectral coverage of photorefractive holography using semi-insulating MQW devices.

High-speed 3D imaging using photorefractive holography

We present a rapid whole-field, 3-D imaging technique based on low coherence interferometry using photorefractive holography in semi-insulating Multiple Quantum Well (MQW) devices, which is capable of whole-field depth resolved 3-D imaging at frame rates exceeding 475 fps. Photorefractive holography provides a unique mechanism to discriminate against a diffuse light background, making it attractive for imaging through turbid media, e.g. for biomedical applications. We note that this whole-field technique can exploit sources of almost arbitrary spatial coherence, including LEDs, fibre-coupled laser diode arrays, broadband c.w. lasers etc, as well as ultrafast laser pulses. The use of spatially incoherent light greatly reduces the deleterious impact of speckle.

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.