J. McGinty

High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging

We report the development of a high-speed wide-field fluorescence-lifetime imaging (FLIM) system that provides fluorescence-lifetime images at rates of as many as 29 frames/s. A FLIM multiwell plate reader and a potentially portable FLIM endoscopic system operating at 355-nm excitation have been demonstrated.

High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events

We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.

Toward the clinical application of time-domain fluorescence lifetime imaging

High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.

Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier

High-speed (video-rate) fluorescence lifetime imaging (FLIM) is reported using two different time-domain approaches based on gated optical image intensifier technology. The first approach utilizes a rapidly switchable variable delay generator with sequential image acquisition, while the second employs a novel segmented gated optical imager to acquire lifetime maps in a single shot. Lifetimes are fitted using both a non-linear least-squares fit analysis and the rapid lifetime determination method. Monte Carlo simulations were used to optimize the acquisition parameters and a comparison between theory and experiment is presented. The importance of single-shot imaging to minimize the deleterious impact of sample movements is highlighted. Real-time FLIM movies of multi-well plate samples and tissue autofluorescence are presented.

Signal-to-noise characterization of time-gated intensifiers used for wide-field time-domain FLIM

Time-gated imaging using gated optical intensifiers provides a means to realize high speed fluorescence lifetime imaging (FLIM) for the study of fast events and for high throughput imaging. We present a signal-to-noise characterization of CCD-coupled micro-channel plate gated intensifiers used with this technique and determine the optimal acquisition parameters (intensifier gain voltage, CCD integration time and frame averaging) for measuring mono-exponential fluorescence lifetimes in the shortest image acquisition time for a given signal flux. We explore the use of unequal CCD integration times for different gate delays and show that this can improve the lifetime accuracy for a given total acquisition time.

An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

We demonstrate that spectral selection from a supercontinuum generated in a microstructured fibre can provide a continuously electronically tunable ultrafast spatially coherent source for confocal microscopy and both scanning and wide field fluorescence lifetime imaging.

Fluorescence lifetime imaging for biomedicine

I will review our development and application of fluorescence lifetime imaging implemented in microscopy, tomography and endoscopy to provide molecular readouts across the scales from super-resolved microscopy through imaging of disease models to clinical applications.

An electronically tunable ultrafast laser source applied to fluorescence imaging and microscopy including fluorescence lifetime imaging

We demonstrate a continuously electronically tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated by injecting infrared femtosecond pulses in to a microstructured fibre. We demonstrate this source applied to confocal and wide-field microscopy, as well as multiwell-plate imaging. We also report first application of a tunable supercontinuum source (TSS) to FLIM and to in situ measurements of relative excitation spectra. We are currently applying this source to hyperspectral imaging and working towards a system for acquiring the full fluorescence excitation-emission matrix. Fluorescence lifetime imaging (FLIM) can provide further information about fluorescent samples and requires a rapidly modulated or pulsed light source, in general, (tunable) ultrafast light sources, particularly in the visible, are complex and expensive and usually require a significant degree of adjustment to achieve tunability

Video rate fluorescence lifetime imaging and fluorescence lifetime endoscopy

We report real-time (video-rate) fluorescence lifetime imaging and its application to tissue autofluorescence and endoscopy, demonstrating FLIM of unstained ex vivo tissue at update rates of 5.5Hz through a flexible endoscope.