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.
Multi-beam confocal sectioned fluorescence lifetime imaging microscopy is demonstrated using a Yokogawa spinning disk. The single-photon excitation source is a supercontinuum generated from a Ti:sapphire seeded photonic crystalline fibre.
Tissue contains many natural fluorophores and therefore by exploiting autofluorescence, we can obtain information
from tissue with less interference than conventional histological techniques. However, conventional intensity imaging is
prone to artifacts since it is an absolute measurement. Fluorescence lifetime and spectral measurements are relative
measurements and therefore allow for better measurements. We have applied FLIM and hyperspectral FLIM to the
study of articular cartilage and its disease arthritis. We have analyzed normal human articular cartilage and cartilage
which was in the early stages of disease. In this case, it was found that FLIM was able to detect changes in the diseased
tissue that were not detectable with the conventional diagnosis. Specifically, the fluorescence lifetimes (FL) of the cells
were different between the two samples. We have also applied hyperspectral FLIM to degraded cartilage through
treatment with interleukin-1. In this case, it was found that there was a shift in the emission spectrum with treatment and
that the lifetime had also increased. We also showed that there was greater contrast between the cells and the
extracellular matrix (ECM) at longer wavelengths.
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.