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.
Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the
diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH.
This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo
state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite
autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires
fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting
and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and
binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis
between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.
To aid the in vivo diagnosis of skin lesions, we present the design and implementation of a 4 channel FLIM
detector and a hyperspectral imaging detector into a clinically licensed commercial two-photon tomograph. We
have also implemented image segmentation algorithms to facilitate the automated processing of the large
volumes of data produced. The first detector is based on multispectral time correlated single photon counting,
providing four channel fluorescence lifetime images. The second detector is a prism-based CCD hyperspectral
imager. These detectors provide the capability to extract the relative content and state of autofluorescence
compounds present in biological tissue.
We describe an optically-sectioned FLIM multiwell plate reader that combines Nipkow microscopy with
wide-field time-gated FLIM, and its application to high content analysis of FRET. The system acquires
sectioned FLIM images in <10 s/well, requiring only ~11 minutes to read a 96 well plate of live cells
expressing fluorescent protein. It has been applied to study the formation of immature HIV virus like
particles (VLPs) in live cells by monitoring Gag-Gag protein interactions using FLIM FRET of HIV-1 Gag
transfected with CFP or YFP. VLP formation results in FRET between closely packed Gag proteins, as
confirmed by our FLIM analysis that includes automatic image segmentation.
Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy
(TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system
(DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time
correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum.
Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis
of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.
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.
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.
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