We present the design, characterization and application of a novel, rapid, optically sectioned hyperspectral fluorescence
lifetime imaging (FLIM) microscope. The system is based on a line scanning confocal configuration and uses a highspeed
time-gated detector to extract lifetime information from many pixels in parallel. This allows the full spectraltemporal
profiles of a fluorescence decay to be obtained from every pixel in an image. Line illumination and slit
detection also gives the microscope a confocal optical sectioning ability. The system is applied to test samples and
unstained biological tissue. In future, this microscope will be combined with recently-developed continuously
electronically tunable, pulsed light sources based on tapered, micro-structured optical fibers. This will allow
hyperspectral FLIM to be combined with the advantages of excitation spectroscopy to gain further insight into complex
biological specimens including tissue and live cell imaging.
The application of autofluorescence in non-invasive medical diagnostics could have great potential. Two major
drawbacks inherent to this approach are low signal levels compared to those from exogenous fluorescent probes
and complexity caused by the multiplicity of fluorescent biomolecules in tissue. Here we present a new optical
system that is based on single channel detection via an optical fiber and can measure the fluorescence emission
spectrum and fluorescence lifetime simultaneously for excitation wavelengths of 355 and 435nm. Single channel
measurements integrate the signal normally available in an imaging setup and therefore have a better signal-tonoise
ratio. Resolving both the fluorescence emission spectrum and fluorescence lifetime provides the opportunity
to discriminate multiple fluorophores. This instrument is intended for NAD(P)H and flavin measurements for
the dynamic monitoring of cellular metabolism and optical measurements of cancerous tissue. Initial results from
a study of live cells and a clinical study of human skin lesions are presented.
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.
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.