The current paper describes the design and population testing of a flicker sensitivity assessment technique corresponding to the psychophysical approach for straylight measurement. The purpose is twofold: to check the subjects' capability to perform the straylight test and as a test for retinal integrity for other purposes. The test was implemented in the Oculus C-Quant straylight meter, using homemade software (MATLAB). The geometry of the visual field lay-out was identical, as was the subjects' 2AFC task. A comparable reliability criterion ("unc") was developed. Outcome measure was logTCS (temporal contrast sensitivity). The population test was performed in science fair settings on about 400 subjects. Moreover, 2 subjects underwent extensive tests to check whether optical defects, mimicked with trial lenses and scatter filters, affected the TCS outcome. Repeated measures standard deviation was 0.11 log units for the reference population. Normal values for logTCS were around 2 (threshold 1%) with some dependence on age (range 6 to 85 years). The test outcome did not change upon a tenfold (optical) deterioration in visual acuity or straylight. The test has adequate precision for checking a subject's capability to perform straylight assessment. The unc reliability criterion ensures sufficient precision, also for assessment of retinal sensitivity loss.
In this study, a ratiometric quantification method is developed and applied to monitor mesotetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method
employs a combination of dual-wavelength excitation and dual-wavelength detection. The excitation and
detection wavelengths were selected in close to NIR. The first excitation wavelength was used to excite the
mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be substracted.
Subsequently the difference was divided by the autofluorescence. Since the method applies division of signal
with no mTHPC fluorescence, theory suggests on linear dependency of the method on photosensitizer
concentration.
Photodynamic therapy (PDT) using protoporpyrin IX (PpIX) precursors like 5-aminolevulinic acid (ALA) or
methyl-aminolevulinate (MAL) has shown to be effective in the treatment of various skin diseases. Using ALA
we have shown in numerous studies a significantly improved efficacy by applying light fractionation with a
long dark interval. In contrast, in the hairless mouse model, the PDT efficacy using MAL is unaffected by
adopting this approach. More acute edema is found after ALA-PDT suggesting a difference in response of
endothelial cells to PDT.
To investigate the role of endothelial cells, cryo-sections of hairless mouse skin after 4 hours of topical MAL
or ALA application were stained with a fluorescent endothelial cell marker (CD31). Co-localization of this
marker with the PpIX fluorescence was performed using the spectral imaging function of the confocal
microscope. We have also used intra-vital confocal microscopy to image the PpIX fluorescence distribution in
correlation with the vasculature of live mouse skin.
Our results show PpIX fluorescence at depth in cryo-sections of mouse skin after 4 hours of topical
application. Co-localization has shown to be difficult due to the changes in tissue organization caused by the
staining procedure. As expected we found high PpIX fluorescence levels in the epidermis after both MAL and
ALA application using intra-vital microscopy. After ALA application more PpIX fluorescence was found deep in
the dermal layer of the skin than after MAL. Furthermore we detected localized fluorescence in unidentified
structures that could not be correlated to blood vessels or nerves.
We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan®) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg/kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; ~0.2 mm3 and ~102 mm3, respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.
We have developed a new technique, fluorescence differential path length spectroscopy (FDPS), that enables the quantitative investigation of fluorophores in turbid media. FDPS measurements are made with the same probe geometry as differential path length spectroscopy (DPS) measurements. Phantom measurements are performed for two fiber diameters (400 µm and 800 µm) and for a wide range of optical properties (µ: 0 to 10 mm−1; µa: 0 to 2 mm−1) to investigate the influence of the optical properties on the measured differential fluorescence signal. The differential fluorescence signal varies by a factor of 1.4 and 2.2 over the biologically relevant scattering range (0.5 to 5 mm−1) for a given fluorophore concentration for 400 µm and 800 µm fibers, respectively. The differential fluorescence signal is attenuated due to absorption at the excitation wavelength following Lambert-Beer's law with a path length equal to the differential path length.
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