SignificanceThe clinical use of optical methods for in vivo skin imaging is limited by skin strong scattering properties, which reduce image contrast and probing depth. The efficiency of optical methods can be improved by optical clearing (OC). However, for the use of OC agents (OCAs) in a clinical setting, compliance with acceptable non-toxic concentrations is required.AimOC of in vivo human skin, combined with physical and chemical methods to enhance skin permeability to OCAs, was performed to determine the clearing-effectiveness of biocompatible OCAs using line-field confocal optical coherence tomography (LC-OCT) imaging.ApproachNine types of OCAs mixtures were used in association with dermabrasion and sonophoresis for OC protocol on three volunteers hand skin. From 3D images obtained every 5 min for 40 min, the intensity and contrast parameters were extracted to assess their changes during the clearing process and evaluate each OCAs mixture’s clearing efficacy.ResultsThe LC-OCT images average intensity and contrast increased over the entire skin depth with all OCAs. The best image contrast and intensity improvement was observed using the polyethylene glycol, oleic acid, and propylene glycol mixture.ConclusionsComplex OCAs featuring reduced component concentrations that meet drug regulation-established biocompatibility requirements were developed and proved to induce significant skin tissues clearing. By allowing deeper observations and higher contrast, such OCAs in combination with physical and chemical permeation enhancers may improve LC-OCT diagnostic efficacy.
The clinical use of optical methods for in vivo skin imaging is limited by skin strong scattering properties, which reduce image contrast and probing depth. The efficiency of optical methods can be improved by optical clearing (OC). However, for the use of optical clearing agents (OCAs) in a clinical setting, compliance with acceptable non-toxic concentrations is required. Optical clearing of in vivo human skin, combined with physical and chemical methods to enhance skin permeability to OCAs, was performed to determine the clearing-effectiveness of biocompatible OCAs using Line-field Confocal Optical Coherence Tomography (LC-OCT) imaging. Nine types of OCAs mixtures were used in association with dermabrasion and sonophoresis for optical clearing protocol on three volunteers hand skin. From 3D images obtained every five minutes for 40 minutes, the intensity and contrast parameters were extracted to assess their changes during the clearing process and evaluate each OCAs mixture’s clearing efficacy. LC-OCT images average intensity and contrast increased over the entire skin depth with all OCAs. The best image contrast and intensity improvement was observed using the Polyethylene Glycol, Oleic Acid and Propylene Glycol mixture. Complex OCAs featuring reduced component. concentrations that meet drug regulation-established biocompatibility requirements were developed and proved to induce. significant skin tissues clearing. By allowing deeper observations and higher contrast, such OCAs in combination with physical and chemical permeation enhancers may improve LC-OCT diagnostic efficacy.
Skin optical clearing is the method providing the increase of the depth and the contrast of non-invasive optical techniques. Dehydration is one of the possible mechanisms of optical clearing that may lead to reduce in both skin thickness and backscattering. However, the contribution of dehydration of skin to the collimated transmittance spectra is not studied in details. This paper presents the results of such study with rat skin ex vivo. Dehydration of skin sample was provided using a fan with a heater. Throughout the experiment, a warm air flux (~ 36 °C) was directed to the dermal side of each sample. Transmittance spectra were obtained using a system of two optical fibers equipped with collimators in the wavelength range of 400-800 nm. The thickness was measured during dehydration using a micrometer. As a result, it was found that a decrease in the thickness of the skin sample during dehydration led to an increased light transmission through the skin (an 11-fold increase at λ= 700 nm), and, at the same time, to an increase in the light attenuation coefficient μt due to an increase in the concentration of scatterers in the skin per unit volume.
Skin cancers including carcinomas and melanomas are currently the most common cancers in fair skinned humans. Histopathology, requiring invasive tissue biopsy and processing, is the gold standard for cancer diagnosis. Optical Coherence Tomography (OCT) has emerged as a label-free, non-traumatic and non-invasive method that can be used in vivo to image skin tissues (from stratum corneum to dermis) and therefore contribute to skin cancer diagnosis. Some of the major limitations of OCT imaging techniques are a lower resolution compared to histology and a limited penetration depth due to skin tissues’ strong optical scattering. Optical clearing has been investigated for several years as one of the solutions to overcome these problems by inducing a reversible decreased scattering and thus allowing a better contrast and an improved light penetration depth within biological tissues. Clearing is achieved using optical clearing agents (OCA) combined with chemical enhancers (used to better pass through the stratum corneum layer). As a first step, the current study aims at defining the quantitative features (intensity profile, image statistics, texture descriptors) that are best suited to quantify optical clarification kinetics from images acquired using Linefield Confocal-OCT (LC-OCT) device. This will help analyzing the relationship between visible optical clearing and OCT devices resolution.
The use of optical clearing agents makes it possible to increase the probing depth of non-invasive optical diagnostic methods. However, the protective epidermal barrier of the skin prevents the penetration of hydrophilic immersion liquids into the deeper layers of the skin. The aim of this study is to improve the efficiency of skin optical clearing by increasing the permeability of the epidermis for polyethylene glycol with MW 300 (PEG-300) using different physical approaches.
We present the results of a comparative analysis of optical immersion clearing of skin in laboratory rats in vivo with and without preliminary ablation of epidermis, fraction microablation and sonophoresis. Laser ablation and microablation has been implemented using a setup based on a pulsed erbium laser (λ= 2940 nm) with different hand-pieces. Sonophoresis has been applied during 2 min with the following parameters: 1 MHz, 2 W/cm2, continuous mode. As an optical clearing agent (OCA) polyethylene glycol (PEG-300) has been used. Basing on optical coherence tomography, we have estimated the attenuation coefficient in the process of optical clearing in two regions at depths of 50–170 µm and 150–400 µm.
The results have shown that both surface ablation of skin in vivo lead to the local edema of the affected region that increases the scattering coefficient. However, the intense evaporation of water from the ablation zone facilitates the optical clearing at the expense of tissue dehydration, particularly in the upper layers. Besides, fractional ablation enhances the both rate and degree of optical clearing in upper dermis layers but not affects the deeper dermis. Sonophoresis provides the most effective penetration of OCA into deeper dermis. Time dependences of attenuation coefficient in two regions of dermis at the separate and combined laser and ultrasound impact on the epidermal permeability for PEG-300 have been obtained.
Optical methods in medicine are exploit the light propagation through the biological tissue, to non-invasively extract morphological and geometrical parameters of the tissue under investigation, from the detected light. However, because the transport of photons into biological tissues of interest is weakened due to losses caused by multiple light scattering, absorption and reflectance, the depth resolution and photon penetration depth of these optical methods still need to be improved. The optical properties of biological tissues can be controlled using special biocompatible hyperosmotic agents or Optical Clearing Agents (OCA). The latter might penetrate inside a tissue, interact with and modify its natural high-scattering formation through different ways and thus, increase the photons penetration within the biological tissue. The aim of this work was to study the modifications in depth resolution of the proposed Spatially Resolved Multi-Modal Spectroscopy (SRMMS) method by combining it with an optical clearing approach and to estimate the applicability of this method for future clinical use.
As an OCA, PEG-400/DMSO (80%/20% vol/vol) and Sucrose/PEG-400/PG (50%/45%/5% vol/vol) solutions were used. Depending on the experimental condition, either one of 2 OCA, or saline solution or nothing ("dry" condition) was applied on the skin surface of 2-layer hybrid model, made of skin layer on top and the gel layer, containing exogenous fluorophore Chlorin e6, on bot.
The SRMMS probe made of one central excitation fiber and 4 rings of collecting fibers for collecting spectroscopic data with the separation by depth, was placed in contact with the skin at the area of application. Then, Diffuse Reflectance and excited on a different wavelengths (365,385,395, 405 and 415 nm) Autofluorescence spectra were aquired at initial time point, and then every 4 minutes within 36 minutes. Aquired DR and AF spectra were preprocessed, and the Area Under the Curve (AUC) values were calculated for the specific wavelength bandwidths of interest for the skin-AF and Ce6-fluorescence spectra obtained at every of the 5 excitation peaks. AUC values were normalized to the T0 respective values to get the time kinetics curves of the spectroscopic signals.
Time kinetics of skin-AF and Ce6-fluorescence spectrum AUC showed that under 36 minutes of OCA application it is possible to observe certain enhanced light penetration effect through an increasing of normalized Ce6 AUC kinetics and a decreasing of normalized AF AUC kinetics. Thus, it was possible to observe that optical clearing led to increased depth resolution with time. Moreover, the effect of clearing itself was observed to be dependent on the chosen OCA, while the saline solution application didn’t cause significant changes in normalized fluorescence intensities. The result of “dry” condition experiment highlighted that while there were no significant changes in AF spectrum amplitudes, a strong increase of Ce6 fluorescence emission intensity was collected from the bottom gel layer. This is probably due to drying of an ex vivo skin sample and the applied probe pressure effect.
Optical clearing of the human skin under the action of hyaluronic acid (HA) as an enhancer for optical clearing by polyethylene glycol (PEG-300) was studied in vivo by optical coherent tomography (OCT) method on the wavelength 930±5 nm. The results have showed an increase in the transparency of the epidermis at the optical depth of 50±5 μm by 44±9% within 15 min with the use of a solution of HA in combination with sonophoresis before application of PEG-300 to the skin surface. At the same time, when using PEG-300 without preliminary treatment of the skin with the HA, an increase in the transparency of the epidermis at the same depth was 21±8% within 25 min. In dermis at the depth of 500 μm the OCT signal in the experimental group increased more than 1.6 fold what indicates an increasing of optical probing depth.
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