The COVID-19 pandemic increased interest in large-scale disinfection of public spaces using UV-C germicidal lights. However, excessive UV-C exposure harms the skin and cornea. Although novel Far UV-C (180 – 230 nm) sources are widely considered safe, an easy and non-invasive experimental assessment of UV damage could help to exclude any health risks of extensive Far UV-C exposure.
Dynamic-microscopic optical coherence tomography (dmOCT) is a non-invasive, label-free technique providing sub-cellular resolution images. This study used dmOCT to confirm Far UV-C's skin safety with in-vitro models. Results showed no signs of keratinocyte damage, while UV-B exposed samples express immediate structural and functional changes.
This project proposes an alternate method to DIN/ISO parameters for analyzing the effects of intrinsic and extrinsic aging on skin morphology. This method improves the efficacy of assessing aging by evaluating the surface and subsurface of the skin. The volumetric data obtained using OCT was processed using a machine learning approach to obtain DEJ segmentation, which was further segmented to obtain the morphological information of the micro-plateaus. The results showed that the flattening of DEJ due to aging is accompanied by changes in the size and shape of micro-plateaus in the DEJ.
Large area fractional laser treatment (LAFLT) is a new fat removal and weight loss concept, which applies microscopic thermal injuries to large parts of the skin. These injuries induce a controlled metabolic response which temporarily increases the energy expenditure (EE), thereby reducing overall weight. In a mouse model, we investigated the dosimetry settings to induce this metabolic reaction. Therefore, mice were housed in metabolic cages and their body composition was analyzed. Results showed a significant EE increase (31% - 91%, p < 0.05) over a 6-day period which seems to suggest that an enhanced metabolic state can be achieved.
An oxygen-sensing microneedle array (MNA) was developed to monitor oxygen partial pressure in tissue in a minimally invasive fashion. The working principle is based on the quenching of emission intensity and phosphorescence lifetime of a Pt-core porphyrin embedded into the MNA. It was shown that the MNA is sufficiently robust to puncture human skin and to detect changes in oxygenation within the physiologically relevant range of 0-160 mmHg. Moreover, it was demonstrated that the MNA can be implemented into a wearable wireless optical readout system rendering the MNA a novel and user-friendly technique to monitor oxygen partial pressure in tissue.
Ablative fractional laser (AFL) treatment prior to application of a topical drug enhances drug uptake and the resultant coagulation zone (CZ) surrounding each fractional photothermal injury may provide a reservoir for sustained drug release. In this work, we evaluate how morphological changes in the skin after AFL affect the uptake of an intrinsically fluorescent topical antibiotic. Brightfield images of NBTC stained histopathological slides were evaluated using a deep learning approach for semantic segmentation of fractional laser patterns for automatic assessment of laser hole diameter and CZ morphology. Last, collagen denaturation and drug uptake were quantified via polarization and fluorescence microscopy, respectively.
KEYWORDS: Optical coherence tomography, Skin, In vitro testing, Temperature metrology, Mode conditioning cables, Signal processing, Scattering, Acquisition tracking and pointing, 3D modeling, Motion models
Microscopic optical coherence tomography (OCT) provides three-dimensional, high-resolution imaging but lacks (sub-) cellular contrast. Dynamic-microscopic OCT (dmOCT) is an approach exploiting dynamic changes of the scattering behavior in metabolically active cells. However, the underlying cellular processes responsible for those intensity fluctuations and hence the dynamic signals are not finally identified yet. Here, we present the effects of different temperatures and metabolic reagents on dmOCT images of an in-vitro human skin model. Our data indicates a dependency of the dmOCT signals on metabolic activity rather than Brownian motion and suggests dependency on the metabolic state.
Intrinsic and extrinsic aging of human skin induces significant morphological changes to its surface. The most prominent and important feature in cosmetics and dermatology is the alteration of the wrinkles. Roughness parameters (Ra, Rmax) described by DIN/ISO disregard the skin’s micro-structure. Hence, we introduce an alternative method of skin roughness evaluation by analyzing the size and shape of micro-structures using optical coherence tomography. Measurements of young and elderly subjects were acquired. The skin of elderly subjects showed a decrease in micro-structures compared to the skin of young subjects which was predominated by triangular shapes, whereas rhomboids prevail among the elderly.
Optical coherence tomography angiography (OCTA) provides in-vivo images of microvasculature. In skin it often represents a dynamic perfusion state without depicting the actual extent of the vascular network. Here, we present the capillary refill method for obtaining a more accurate anatomic representation of surface capillary networks in human skin using OCTA.
OCTA images were captured at baseline displaying ambient capillary perfusion and after compression and release of the skin representing the network of existing capillaries at full capacity. This method provides mapping of cutaneous capillary networks independent of ambient perfusion comparable to histological analysis of biopsies on identical skin sites.
Cryolipolysis has become a popular non-invasive method of reducing excess fat by cooling. However, the results vary as metabolism, gender and diet may affect the freezing point of human subcutaneous fat. To increase the success for all patients, it is essential to better understand the process of cryolipolysis in vivo. Therefore, we have developed a side-facing needle probe with an outer diameter of 390 μm achieving a lateral resolution of 10 μm at a working distance of 1.5 mm. To obtain a spatially resolved visualization of the immediate processes involve in cryolipolysis, cross-sectional images was obtained by moving the needle probe back and forth in a transparent catheter. At the tip, the transparent catheter was equipped with a lancet for smoothly penetrating through the skin. By a rigorous design including optical wave simulation and by a careful combination of different materials astigmatic aberrations were avoided. Ex vivo measurements on subcutaneous porcine fat were performed to confirm, that imaging with the needle probe is a suitable method for investigating phase changes.
Optical coherence tomography angiography (OCTA) provides in-vivo images of microvascular perfusion in high resolution. For its application to basic and clinical research, an automatic and robust quantification of the capillary architecture is mandatory. Only this makes it possible to reliably analyze large amounts of image data, to establish biomarkers, and to monitor disease developments. However, due to its optical properties, OCTA images of skin often suffer from a poor signal-to-noise ratio and contain imaging artifacts. Previous work on automatic vessel segmentation in OCTA mostly focuses on retinal and cerebral vasculature. Its applicability to skin and, furthermore, its robustness against imaging artifacts had not been systematically evaluated. We propose a segmentation method that improves the quality of vascular quantification in OCTA images even if corrupted by imaging artifacts. Both the combination of image processing methods and the choice of their parameters are systematically optimized to match the manual labeling of an expert for OCTA images of skin. The efficacy of this optimization-based vessel segmentation is further demonstrated on sample images as well as by a reduced error of derived quantitative vascular network characteristics.
The evaluation of complex metabolic changes of individual live cells and heterogeneous cell cultures is not feasible using traditional methods due to their destructive behavior and lack of spatial information. Two-photon excited fluorescence of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) facilitate a label-free and non-destructive evaluation of metabolic activity. This study explores the phasor approach in combination with two-photon fluorescence lifetime imaging microscopy (FLIM) as a potential method to evaluate pharmacologically induced metabolic changes that occur during the browning of adipocytes. The possibility of browning of white adipose cells is a desirable prospect for the treatment of obesity and related disorders. Here, we compared the results obtained by Fourier-based phasor analysis with the traditional exponential FLIM analysis as well as results of an extracellular flux analyzer. The alteration of glycolytic function and oxidative phosphorylation after treatment with pharmacological reagents significantly shift the contribution of each of the fluorescence lifetime components to the total fluorescence intensity. Further, we showed that the ratios of the lifetime components obtained by the phasor approach reflect the shift in mitochondrial and cytosolic NADH concentration. The phasor analysis agrees with traditional assessments, such as the optical free-to-bound NADH ratio as well as the oxygen consumption rate and extracellular acidification rate as determined by the extracellular flux analyzer. Our results support the concept that non-invasive sensing of fat metabolism and browning of fat may be possible by analyzing the fluorescence lifetime of NADH using the phasor approach.
The ability to image the physiology of microvasculature with high spatial resolution in three dimensions
while investigating structural changes of skin, is essential for understanding the complex
processes of skin aging, wound healing and disease development. Further, the quantitative, automatic
assessment of these changes enables to analyze large amounts of image data in an abstract
but comprehensive manner.
However, previous work using OCT with methods of angiography was imaging less scattering,
hence more challenging tissue than skin, such as brain and retina tissue. The published methods
for capillary segmentation were mostly non-automatic, poorly benchmarked against state-of-the-art methods of computer vision and not applied to investigate medical processes and studies in a comprehensive fashion.
Here, segmentation of capillaries in skin is reported and its efficacy is demonstrated in both, a
longitudinal mouse study and a preliminary study in humans. By combining state-of-the-art image
processing methods in an optimized way, we were able to improve the segmentation results and analyze the impact of each post-processing step.
Furthermore, this automatic segmentation enabled us to analyze big amounts of
datasets automatically and derive meaningful conclusions for the planning of clinical studies.
With this work, optical coherence tomography is combined with methods of computer vision to a diagnostic
tool with unique capabilities to characterize vascular diversity and provide extraordinary
opportunities for dermatological investigation in both, clinics and research.
Cryolipolysis is a well-established cosmetic procedure for non-invasive local fat reduction. This technique selectively destroys subcutaneous fat cells using controlled cooling. Thickness measurements of subcutaneous fat were conducted using a mouse model. For detailed examination of mouse skin optical coherence tomography (OCT) was performed, which is a non-invasive imaging modality. Due to a high number of image slices manual delineation is not feasible. Therefore, automatic segmentation algorithms are required. In this work an algorithm for the automatic 3D segmentation of the subcutaneous fat layer is presented, which is based on a random forest classification followed by a graph-based refinement step. Our approach is able to accurately segment the subcutaneous fat layer with an overall average symmetric surface distance of 11.80±6.05 μm and Dice coefficient of 0.921 ± 0.045. Furthermore, it was shown that the graph-based refining step leads to increased accuracy and robustness of the segmentation results of the random forest classifier.
Traditional assessments of cellular metabolism are often destructive, time consuming and without visual information. Fluorescence lifetime imaging microscopy (FLIM) provides a highly sensitive, non-invasive, and label-free alternative.
This study uses FLIM in combination with two-photon microscopy to investigate pharmacological induced metabolic changes of adipocytes via changes in the fluorescence of the metabolic co-factors NADH and FAD. In agreement with recent publications NADH fluorescence suggests the presence of four distinct lifetimes in cell culture and tissue with two unbound and two protein bound states which show different responses to treatment with metabolic modifiers. We evaluated the effects on NADH fluorescence lifetime after systematic manipulations to change the balance between oxidative and glycolytic metabolism using five pharmacological reagents - Oligomycin, 2-DG, FCCP, Rotenone, and Glucose - which interact with different parts of the metabolic pathway. We established several ratios between the four distinct lifetimes of NADH after treatment and compared the results to oxygen consumption rate and extracellular acidification rate.
We demonstrated, for the first time, a correlation between the two unbound fluorescence lifetimes components and glycolytic and oxidative metabolic activity with a significant higher sensitivity compared to the commonly used free-to-bound ratio of NADH. Analyzing all four lifetime components of NADH has the potential to become a powerful tool to evaluate metabolic activity of adipocytes with subcellular resolution.
In dermatology the reflexes of vasoconstriction and vasodilation are known as important mechanisms of thermoregulation of the inner body. Imaging the physiology of microvasculature of the skin with high spatial resolution in three dimensions while reacting to changes in temperature is crucial for understanding the complex processes of vasodynamics, which result in constriction and dilation of vessels. However, previous studies using Laser-Doppler flowmetry and -imaging could not provide reliable angiographic images which allow to quantify changes in blood vessel diameter. Here, we report a different approach for angiographic imaging of microvasculature of a anaesthetized rodent model using speckle variance optical coherence tomography (svOCT) during and after localized cooling. Therefore a commercial OCT with a center wavelength of 1.3 μm and a spatial resolution of 13µm was used in combination with a custom built cooling device to image such reflexes at the mouse ear pinna and dorsal skinfold. Cooling was applied in steps of 2−5◦ C starting at the baseline temperature of 27◦ C down to −10◦ C.
To our surprise and in contrast to the general opinion in literature, we were able to observe that the majority of vessels with a diameter larger than 20 μm maintain perfused with a constant diameter when the tissue is cooled from baseline to subzero temperatures. However, vasoconstriction was observed very rarely and only in veins, which led to their occlusion. The results of this experiment lead us to reconsider essential aspects of previous understanding of temperature-induced vasodynamics in cutaneous microvasculature.
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