Degradation of articular cartilage extracellular matrix (ECM) by proteolytic enzyme is the hallmark of arthritis that leads to joint destruction. Detection of early biochemical changes in cartilage before irreversible structural damages become apparent is highly desirable. Here we report that the autofluorescence decay profile of cartilage is significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A multidimensional fluorometer utilizing ultraviolet excitation at 355 nm or 375 nm coupled to a fibreoptic probe was developed for single point time-resolved AFL measurements of porcine articular cartilage explants treated with different proteinases. Degradation of cartilage matrix components by treating with bacterial collagenase, matrix metalloproteinase 1, or trypsin resulted in significant reduction of AFL of the cartilage in both a dose and time dependent manner. Differences in cartilage AFL were also confirmed by fluorescence lifetime imaging microscopy (FLIM). Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may be utilized for diagnosis of arthritis as well as monitoring the efficacy of anti-arthritic therapeutic agents.
We present the application of two fibre-optic-coupled time-resolved spectrofluorometers and a compact steady-state
diffuse reflected light/fluorescence spectrometer to in vivo and ex vivo studies of skin cancer and osteoarthritis. In a
clinical study of skin cancer, 27 lesions on 25 patients were investigated in vivo before surgical excision of the region
measured. Preliminary analysis reveals a statistically significant decrease in the autofluorescence lifetime of basal cell
carcinomas compared to neighbouring healthy tissue. A study of autofluorescence signals associated with the onset of
osteoarthritis indicates autofluorescence lifetime changes associated with collagen degradation.
We have developed a rapid microfluidic mixing device to image fast kinetics. To verify the performance of the device it
was simulated using computational fluid dynamics (CFD) and the results were directly compared to experimental
fluorescence lifetime imaging (FLIM) measurements. The theoretical and measured mixing times of the device were
found to be in agreement over a range of flow rates. This mixing device is being developed with the aim of analysing fast
enzyme kinetics in the sub-millisecond time domain, which cannot be achieved with conventional macro-stopped flow
devices. Here we have studied the binding of a DNA repair enzyme, uracil DNA glycosylase (UDG), to a fluorescently
labelled DNA substrate. Bulk phase fluorescence measurements have been used to measure changes on binding: it was
found that the fluorescence lifetime increased along with an increase in the polarisation anisotropy and rotational
correlation time. Analysis of the same reaction in the microfluidic mixer by CFD enabled us to predict the mixing time
of the device to be 46 μs, more than 20 times faster than current stopped-flow techniques. We also demonstrate that it is
possible to image UDG-DNA interactions within the micromixer using the signal changes observed from the
multidimensional spectrofluorometer.
We describe the characterisation of a hyperspectral fluorescence lifetime imaging microscope that exploits high-speed
time-gated imaging technology and a tunable continuum source for 6-D fluorescence imaging. This line-scanning
confocal microscope can record the full spectral-temporal (i.e. excitation-emission-lifetime) fluorescence matrix at each
pixel in a three dimensional (x-y-z) sample. This instrument has been applied to biological samples including model
membranes and live cells labelled with the phase-sensitive membrane dye di-4-ANEPPDHQ, for which significant
variation of lifetime with emission wavelength is observed.
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.
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