Dihydroxyacetone (DHA) has been proposed as a potential alternative to dansyl chloride for use as a fluorescence marker on skin to assess stratum corneum turnover time in vivo. However, the fluorescence from DHA on skin has not been adequately studied. To address this void, a noninvasive, noncontact spectral imaging system is used to characterize the fluorescence spectrum of DHA on skin in vivo and to determine the optimal wavelengths over which to collect the DHA signal that minimizes the contributions from skin autofluorescence. The DHA‐skin fluorescence signal dominates the 580–680 nm region of the visible spectrum when excited with ultraviolet radiation in the 320–400 nm wavelength region (UVA). An explanation of the time‐dependent spectral features is proposed in terms of DHA polymerization and binding to skin.
Dihydroxyacetone (DHA) has been proposed as a potential alternative to dansyl chloride for use as a fluorescence marker on skin to assess stratum corneum turnover time in vivo. However, the fluorescence from DHA on skin has not been adequately studied. To address this void, a noninvasive, noncontact spectral imaging system is used to characterize the fluorescence spectrum of DHA on skin in vivo and to determine the optimal wavelengths over which to collect the DHA signal that minimizes the contributions from skin autofluorescence. The DHA-skin fluorescence signal dominates the 580-680 nm region of the visible spectrum when excited with ultraviolet radiation in the 320-400 nm wavelength region (UVA). An explanation of the time-dependent spectral features is proposed in terms of DHA polymerization and binding to skin.
A spectral microscope is being developed that allows for the identification, localization, and quantification of actives on substrates by providing micrographs with either a reflectance or fluorescence spectrum at each pixel of the image. Such an instrument provides the correlation between the spatial and spectral (i.e., chemical) characteristics of the sample with spatial resolution approaching a few hundred nanometers.Several venders are producing instruments that collect spectra with images. Zeiss, BioRad, and Olympus have instruments that are primarily designed for the collection of confocal images with accompanying fluorescence spectra; these systems are generally geared for biological samples that employ fluorescence tags. Non-microscope venders also sell similar instruments but with limited component options. Our instrument is designed with maximum flexibility for the collection of both reflectance and fluorescence spectra (of varying signal size) on a large variety of samples (from hair, fabrics, paper).The current version of this microscope employs an inverted Zeiss Axiovert 35 in its epi-fluorescence mode for both reflected light and fluorescence. Illumination is provided with a xenon lamp that produces a broad spectrum of light across the entire visible region. The system also has a highpressure mercury vapor lamp when more intensity is needed for UV excitation of fluorescent samples. For fluorescence applications, narrow band (FWHM ~ 5 -10 nm) interference filters are placed in the filter cube to select the desired excitation wavelength. For reflectance applications, these filters are removed. A VariSpec liquid crystal tunable filter (LCTF) from CRI, Inc. is placed between the microscope and the detector and is used to provide the spectral discrimination for both fluorescence and reflectance samples. This device has a 10-nm bandwidth and a wavelength range of 400 -720 nm. The LCTF has the advantage of having fast, random access wavelength selection with no moving parts. It also allows for continuous tuning over hundreds of wavelengths within its operating wavelength range. A Photometrics Quantix with a Peltier-cooled Kodak KAF1401E CCD (1317 x 1035 imaging array, 6.8 x 6.8 µ pixels) from Roper Scientific collects the images. Both the CCD and the LCTF are controlled by Universal Imaging Corporation's MetaMorph software. A diagram of this instrument can be seen in Figure 1. Figure 2 shows the reflectance spectrum of a LabSphere, Inc. Wavelength Calibration Standard (WCS-MC-101) obtained from the spectral microscope (black open squares) and from a Perkin Elmer (PE) Lambda-9 Spectrophotometer with an integrating sphere accessory (red open circles).Each spectrum is plotted on its own axis (spectral microscope on the left and PE data on the right) and the imaging data has been corrected for the overall wavelength-dependent instrument response (light source, CCD and LCTF). The top panel of this figure shows that the overall features of the standard are reproduced very well and the LCTF does an excellent job ...
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