Several cardiovascular disease models for studying plaque rupture have been explored ranging from cell lines to animal models, whereby each one has contributed in different ways to pathology understanding, diagnostics and therapy. However, the scientific community is lacking a reliable animal model of human coronary plaque rupture. The model providing perhaps the highest degree of similarity to the human condition is the swine model that is known to exhibit serious disadvantages such as costly maintenance, time-consuming experiments, lasting several years and very low yield. Hence, we have developed a biomimetic artery modular platform where ex vivo human samples are exposed to an in vivo dynamic environment mimicking blood flow in order to get a rapid and reliable assessment of the intravascular diagnostic tool as well as to perform a multi-parametric screening with light. Our tool offers a unique environment to perform ex vivo dynamic studies such as dynamic biomarkers labelling tests, drug delivery, etc. The platform features computer-controlled experimental conditions and adjustable flow rates across the entire physiological range. Biological samples can be analyzed using traditional microscopy and assays or prepared for more advanced characterization after exposure in the test loop. Contrary to cell line models, the use of real artery samples provides a more comprehensive approach closer to human physiology. Interestingly, this approach allows remarkable time and resource savings compared to existent animal model of plaque vulnerability. Moreover, it offers higher flexibility on the biosample and mitigates related ethical issues.
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 report progress on the construction of an optical sectioning programmable array microscope (PAM) implemented
with a digital micro-mirror device (DMD) spatial light modulator (SLM) utilized for both fluorescence illumination and
detection. The introduction of binary intensity modulation at the focal plane of a microscope objective in a computer
controlled pixilated mode allows the recovery of an optically sectioned image. Illumination patterns can be changed very
quickly, in contrast to static Nipkow disk or aperture correlation implementations, thereby creating an optical system
that can be optimized to the optical specimen in a convenient manner, e.g. for patterned photobleaching, photobleaching
reduction, or spatial superresolution.
We present a third generation (Gen-3) dual path PAM module incorporating the 25 kHz binary frame rate TI 1080p
DMD and a newly developed optical system that offers diffraction limited imaging with compensation of tilt angle
distortion.
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.
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.
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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