KEYWORDS: Phototherapy, Skin, Ultraviolet radiation, Multiphoton microscopy, Monte Carlo methods, Confocal microscopy, Tissues, Reflectivity, Modeling, In vivo imaging
We introduce a “mini-phototest” concept, which reduces the skin area exposed to UV light by 20-folds for skin MED (minimal erythema dose) determination. This significantly minimizes cosmetic effects compared to conventional phototest. We validated this concept by Monte-Carlo simulation, and also studied cellular response induced by mini-size UV exposure using in vivo multimodality microscopy. This novel phototest method may enable site specific light dosimetry, facilitating personalized precision phototherapy.
The key objective of this study is to test the ability of a newly developed laser therapy technique – multiphoton photothermolysis – to generate precise tissue alteration on in vivo human skin, and to demonstrate the efficiency of a prototype multimodal microscopy to monitor skin response in real-time after the laser treatment. Cellular activities such as proliferation, inflammation, exfoliation of injured tissue can be captured over time. This study highlights the potential use of multiphoton photothermolysis for treatment of conditions that preventing collateral damage is critical. Also, multimodal microscopy offers a non-invasive method for monitoring tissue physiology.
Here we propose a novel method to automatically delineate and quantify DEJ zone morphometrics in human skin on a three dimensional (3D) basis in vivo. Quantitative characterization of the DEJ in terms of 3D interdigitation (I), arithmetic mean roughness (Sa), and root mean square roughness (Sq) were calculated. These DEJ features as a function of age were analyzed. The results show age-dependent morphological differences.
KEYWORDS: Skin, In vivo imaging, Microscopy, Ultraviolet radiation, Imaging systems, Confocal microscopy, Two photon imaging, Solar radiation, Biopsy, Biological research
Background: Serial analysis of cellular dynamics over time offers new insights into human skin responses to solar radiation. However, most of the previous studies are based on biopsy ex vivo analysis approaches that preclude the monitoring of the same cells and sites over time. Optical in vivo microscopy enables the possibility of real-time live cell imaging. Here we report a robust non-invasive method to achieve repeated access to the same micro-location over a long period with unprecedented precision.
Methods: The technique is based on a temporary “surface marker” as landmark to help locate the same cells or microstructures between imaging sessions. At baseline, the region-of-interest (ROI) is determined and imaged. At follow up sessions, the ROI can be automatically located. Using this method, we precisely revisited the same cells in human skin after UVB radiation over two weeks. Skin microscopic responses was studied with a multimodality in vivo microscopy system capable of co-registered video rate reflectance confocal microscopy (RCM) imaging, two-photon fluorescence (TPF) imaging and second harmonic generation (SHG) imaging.
Results: The quantitative analysis of TPF signal revealed that melanin distribution pattern changed with time after UVB exposure, suggesting that melanin migrates towards the skin surface. Blood flow was monitored in the same capillary over two weeks. Multimodal analyses enabled accurate calculation of viable epidermis, stratum corneum thickness and cell density variations over time, demonstrating the time points of tissue edema and cell proliferation.
Porphyrins produced by Propionibacterium acnes represent the principal fluorophore associated with acne, and appear as orange-red luminescence under the Wood’s lamp. Assessment of acne based on Wood’s lamp (UV) or visible light illumination is limited by photon penetration depth and has limited sensitivity for earlier stage lesions. Inducing fluorescence with near infrared (NIR) excitation may provide an alternative way to assess porphyrin-related skin disorders. We discovered that under 785 nm CW laser excitation PpIX powder exhibits fluorescence emission in the shorter wavelength range of 600-715 nm with an intensity that is linearly dependent on the excitation power. We attribute this shorter wavelength emission to anti-Stokes fluorescence. Similar anti-Stokes fluorescence was also detected focally in all skin-derived samples containing porphyrins. Regular (Stokes) fluorescence was present under UV and visible light excitation on ex vivo nasal skin and sebum from uninflamed acne, but not on nose surface smears or sebum from inflamed acne. Co-registered CW laser-excited anti-Stokes fluorescence and fs laser-excited multi-photon fluorescence images of PpIX powder showed similar features. In the skin samples because of the anti-Stokes effect, the NIR-induced fluorescence was presumably specific for porphyrins since there appeared to be no anti-Stokes emission signals from other typical skin fluorophores such as lipids, keratins and collagen. Anti-Stokes fluorescence under NIR CW excitation is more sensitive and specific for porphyrin detection than UV- or visible light-excited regular fluorescence and fs laser-excited multi-photon fluorescence. This approach also has higher image contrast compared to NIR fs laser-based multi-photon fluorescence imaging. The anti-Stokes fluorescence of porphyrins within sebum could potentially be applied to detecting and targeting acne lesions for treatment via fluorescence image guidance.
Reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) are non-invasive methods of acquiring morphological images of the skin in vivo. Most research in this area focuses on instruments that are configured for two-dimensional imaging in a horizontal plane parallel to the skin surface. In contrast, conventional histopathologic evaluation of the skin is based on vertical tissue sections that show microscopic features and their interrelationships according to their depth within the skin. The ability to similarly depict the skin in the vertical plane during in vivo microscopic imaging poses several significant challenges with respect to imaging speed, resolution and extractable information. Aiming to address above challenges, we developed a laser scanning multimodal microscopy system which combines RCM and MPM, and has the ability to do fast xz scanning to achieve high resolution vertical “optical sectioning” of in vivo human skin at video rates. RCM and MPM images are obtained simultaneously and co-registered thereby providing complementary morphological information. To validate the performance of this system vertical section RCM and MPM microscopic images of normal human skin in vivo were obtained at half video rates (15 frames/s). Using our system it is possible to discern the following structures: all layers of the epidermis including the stratum lucidum, the dermal-epidermal junction, and the papillary dermis. Blood flow is also visible as evidenced by blood cell movement within vessels. The effective imaging depth is about 200 micrometers. This system provides a means of interrogating human skin noninvasively at an orientation analogous to conventional histological sectioning.
A confocal multiphoton microscopy system with various detection pinholes was used to differentiate backward scattered second harmonic generation (BS-SHG) from backward generated SHG (BG-SHG) based on the fact that BS-SHG is more scattered and therefore has a much bigger spot size than BG-SHG. BS-SHG is quantified from two types of mouse tissues, such as Achilles tendon, and skin, and at various focal depths. It is found that the BS-SHG contributes less to the total backward SHG for the skin than Achilles tendon with thicknesses of around three hundred micrometers. For tissue with larger F/B intensity ratio such as Achilles tendon, increasing the tissue thickness reduces it tremendously. However, for tissue with smaller F/B intensity ratio, tissue thickness increment does not alter it significantly. In addition, larger F/B intensity ratio might be related with a greater scattering coefficient from our Achilles tendon and skin comparison. When the focal point is moved deeper into tissue, the contribution of BS-SHG is found to decrease due to a reduced pass length of the forward propagated photons. On the contrary, when the tissue thickness increases, the contribution of the BS-SHG is increased. These observations for thicker skin tissues are related with our F/B intensity ratio measurement for thin mouse skin sample in terms of that the magnitude of backward generated SHG are dominant among the total backward SHG in mouse skin tissue. Considering the phase mismatching condition in the forward and backward directions, these results may indicate that quasi-phase matching originating from the regular structure of collagen could help with reducing the phase mismatch especially in the backward direction.
The backward second harmonic generation (SHG) in mouse tissues is studied with a confocal multiphoton microscopy system. The total backward collected SHG (B-SHG) consists of the backward generated SHG and the backward-scattered forward-generated SHG (BS-SHG), which can be modeled by a Gaussian and a uniform distribution, respectively, at the confocal pinhole plane. By varying the pinhole size with a series of collection fibers, the proportion of the BS-SHG to the B-SHG and the proportion of BS-SHG to the forward generated SHG can be obtained. The approach is first validated by Monte Carlo simulation. It is then applied to two types of mouse tissues: mouse tail tendon and Achilles tendon. It is found that the BS-SHG contributes less to the B-SHG for the tail tendon than Achilles tendon with thicknesses of ∼300 μm . With the thickness of the Achilles tendon tissue increased to 1000 μm but the focal plane kept at the same depth, as high as ∼10% of the total forward SHG is backscattered and collected. The results indicate that BS-SHG may not be the major source of B-SHG in the tail tendon, but it may be the major source in the Achilles tendon. These methods and results provide a noninvasive method and supporting information for investigating the generation mechanism of SHG and help with optimizing backward SHG microscopy and spectroscopy measurements.
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