S.E.D. Webb

Application of the stretched exponential function to fluorescence lifetime imaging.

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

Imaging the environment of green fluorescent protein.

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.

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.

Studying biological tissue with fluorescence lifetime imaging: microscopy, endoscopy, and complex decay profiles

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.

Time-resolved fluorescence anisotropy imaging applied to live cells

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.

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.

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

Polarization-resolved fluorescence lifetime imaging (FLIM) can provide information about the rotational diffusion of a fluorophore. We have developed a wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) system to quantitatively image both the fluorescence lifetime and the rotational correlation time of a fluorophore. Upon excitation with linearly polarized light, we simultaneously image the parallel and perpendicular polarization components of the fluorescence emission employing a polarization-resolved imager in front of a time-gated intensified CCD. We demonstrate this technique by imaging the rotational correlation time of fluorescein in solutions of varying viscosity in a multiwell plate and in live cells. Introduction

Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser

We report a wide-field fluorescence lifetime imaging (FLIM) system that uses a blue picosecond pulsed diode laser as the excitation source. This represents a significant miniaturization and simplification compared with other time-domain FLIM instruments that should accelerate the development of clinical and real-world applications of FLIM. We have demonstrated this instrument in two configurations: a macroimaging setup applied to multiwell plate assays of chemically and biologically interesting fluorophores and a microscope system that has been applied to imaging of tissue sections. The importance of the adjustable repetition rate of this laser source is discussed with respect to noise reduction and precision in the lifetime determination, illustrating a further significant advantage over conventional mode-locked solid-state lasers.

Biomedical Applications of Fluorescence Lifetime Imaging

Fluorescence lifetime imaging (FLIM) provides a robust functional imaging modality for biomedicine. Recent advances in ultrafast laser and detector technology make FLIM increasingly accessible for life scientists and point the way to new clinical instrumentation.