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17OCT MONITORING OF THE VOLUME FRACTION OF WATER AND OPTICAL CLEARING AGENTS IN
THE SKIN WITH OPTICAL CLEARING EX VIVO
YURIY SURKOV.1,2 ISABELLA SEREBRYAKOVA,1'2 AND ELINA GENINA1,2
'Science Medical Center, Saratov State University, 83 Astrakhanskaya str., 410012 Saratov, Russia
2Laboratory of Laser Molecular Imaging and Machine Learning, Tomsk State University, 36 Lenin av., 634050 Tomsk,
Russia
ABSTRACT
In this work, we implemented a simple method for monitoring the volume fraction of water and an optical clearing agent (OCA) in a skin sample by measuring the group-average refractive index and geometric characteristics of rat skin during its optical clearing (OC) using PEG-300 and glycerol solution by optical coherence tomography (OCT). The main advantage of this OCT monitoring method is that it allows you to simultaneously and continuously measuring both the thickness-averaged refractive index, the geometric thickness, and the volume fractions of water and OCA in the skin sample, without interrupting the natural course of the OC process and without changing the initial position of the sample, which has a positive effect on the measurement accuracy.
Over the years, numerous studies have contributed to a comprehensive study of the skin and provided many instrumental methods for treatment and diagnosis [1, 2]. Such advances have significantly expanded our understanding of the morphological and functional characteristics of the skin and led to the development of various tools that can be used to detect skin diseases at different stages of the pathogenetic link [3-5].
The skin is a multifunctional organ that performs thermoregulatory, receptor, endocrine, metabolic, excretory, respiratory and immune functions. It also helps in the regulation of water loss and metabolism [3]. Changes in skin morphology and impaired skin barrier function may indicate the progression of skin disease. Therefore, early diagnosis of skin diseases, monitoring of the development of the disease and the effectiveness of treatment, as well as age-related changes in the structure of the skin are topical problems in dermatology and cosmetology. In recent decades, a large number of optical methods have been developed, such as confocal Raman microscopy, multiphoton tomography, Raman spectroscopy, laser speckle contrast imaging, optical coherence tomography, which have been successfully used for non-invasive or low-invasive imaging/assessment of the skin [1, 4]. Thus, the development of non-invasive methods for monitoring the properties and condition of the skin is a very promising and relevant area of research.
However, the main limitation of optical diagnostic methods is the low probing depth due to the absorption and scattering of radiation. [5, 6]. To overcome these limitations and increase the depth of penetration of probing radiation into the skin, an optical clearing technique was developed [5, 6], which, in particular, uses various optical clearing agents locally applied to the skin. Among them, for example, glycerol, glucose, polyethylene glycol (PEG), fructose, iohexol, and other agents [5, 6]. OC can significantly increase the depth of probing in the skin, as well as provide better resolution and image quality [5, 6]. To develop the method of immersion optical clearing of biological tissues, it is necessary to know the details of the interaction of immersion liquids with tissue, in particular, the characteristics of osmotic dehydration under the action of OCA, and the process of diffusion of the immersion agent into the tissue [5, 6].
The relevance of the study of the interaction of OCA with biological tissues is due not only to the need to develop methods for optical clearing of tissues, but also to the prevalence of OCA in cosmetics, pharmacological agents, and even electronic cigarettes [7-9]. In addition, new analytical methods are also needed to quickly, inexpensively, and without the involvement of highly qualified operators, obtain a quantitative assessment of the delivery of drugs to the skin with simultaneous visualization of the morphological structure of the tissue [10].
Monitoring of water content in tissues is important for the diagnosis and treatment of various edema, dehydration, burns, and other morpho-degenerative processes of biological tissue, as well as for cosmetic purposes [11]. Despite the fact that the study of skin hydration under various morphological and functional conditions and the measurement of OCA concentration in it are among the main tasks in modern dermatology and cosmetology [4], there is currently no accurate, non-invasive technique for monitoring the water content in tissues with high resolution and sounding depth [4].
Various biophysical methods are widely used to assess the water content of the skin. Existing methods for assessing water in the skin include differential scanning calorimetry, electrical methods, measurement of transepidermal water loss, mechanical methods, photothermal radiometry, spectroscopic methods, confocal Raman microscopy, MRI, etc. [4, 11]. However, these methods have a number of disadvantages: either they do not provide direct non-invasive measurements of the water content in human skin at a depth of more than 100 ^m, or they have a low resolution, or they require complex and time-consuming procedures. In addition, due to the complex nature of the data obtained, advanced methods of data analysis are often required, so the high cost of instruments is a serious limitation, and since most of them are bulky benchtop instruments, measurements are limited to certain anatomical sites [4, 11].
The material for the study was skin samples from the abdominal part of laboratory albino rats with previously removed adipose tissue and hair. After excision of a skin area from the animal's body and removal of hair and subcutaneous adipose tissue from it, the samples were placed in saline for 30 minutes, after which they were removed
from saline. Residual saline was removed from the surface of the sample using filter paper. After that, each sample approximately 1 x 2 cm2 in size was fixed on a smooth and even glass between two glass slides 1.5 mm thick, which were previously placed on a glass substrate. A large amount of OCA was applied to the skin surface and the area next to it, based on the fact that the volume of OCA significantly exceeds the volume of water released into it from the sample, the group refractive index of the solution surrounding the sample can be considered approximately equal to the initial value. After the addition of OCA, the probed area was covered with a cover slip (Fig. 1). This design with the sample was placed in the OCT system so that the edge of the biological tissue and the area where the sample was absent were simultaneously observed. This method of observation is convenient for determining and further analyzing the behavior of the boundaries between the lower surface of the cover slip and the OCA, between the OCA and the upper boundary of the biological tissue, between the biological tissue and the upper boundary of the glass substrate, in addition, the geometric distance between the lower boundary of the cover slip and the upper boundary of the glass substrate remains constant throughout the observation period.
Figure 1: Scheme of sample preparation for OCT probing. 1 - Even and smooth glass substrate; 2 - glass slide 1.5 mm thick;
3 - prepared skin sample; 4 - OCA; 5 - cover glass; region for OCT probing is marked with a red rectangle
The measurement of the group refractive index was carried out using the generally accepted method described in the papers [12-14]. According to this method, it is possible to quantify the average group refractive index of the biological tissue by measuring the optical thickness of the sample and the displacement of the image of the upper border of the slide/substrate in the presence of the sample relative to its position in the absence of the sample. The volume fraction of water and OCA is calculated from the Gladstone Dale ratio [15].
PEG-300 and 40% aqueous glycerol solution were used as OCA. Group refractive index at 930 nm nPEG-300=1.4612, n40„% gl-ol=1.3980, nwater=1.3416 and ndly skm=1.5940 [14].
The region under study with the GAN930V2-BU spectral OCT (Thorlabs, USA), operating at a central wavelength of 930 nm and having a longitudinal and transverse resolution of 5.34 and 7.32 ^m was monitored during 45 minutes. OCT scans were recorded every minute for 10 minutes, then every 5 minutes for least two hours. Thirty A-scans were averaged over time to reduce a speckle noise. The thickness-averaged group refractive index was calculated for each averaged 10 A-scans of the structural skin image and 5 selected regions of interest (ROI).
Figures 2 illustrate typical OCT scans of the skin during its OC with PEG-300 and 40% aqueous glycerol solution. Figures 3 and 4 show the kinetics of changes group refractive index samples - ngr and ks for all ROIs. The group refractive index averaged over the entire probed skin volume for X0 = 930 nm at the initial time was 1.416±0.013, which corresponds with sufficient accuracy to the known data of 1.41±0.03 [13]. 210 minutes after PEG-300 application, ngr in areas remote from the edge (more than 3 mm) averaged 1.484±0.02.
Figure 2: Typical OCT scans of rat skin during its optical clearing at different time intervals after application of (a) PEG-300 and (b) 40% aqueous glycerol solution. Colored rectangles mark areas of interest at a distance from the edge of the sample: 1.
0.67mm; 2. 1.55mm; 3. 2.42mm; 4. 3.30mm; 5.4.17mm
1,56 H 1,541,521,50-g 1,48 -1,461,441,421,40-
ROI 1 ROI 2 ROI 3 ROI 4 ROI 5
1,0 0,90,8! 0,7" 0,60,50,40,3
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100 150 Time, min
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100 150 Time, min
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Figure 3: Time dependence of the (a) average group refractive index and (b) normalized skin thickness during OC with PEG-300
1,46-
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ROI 1 ROI 2 ROI 3 ROI 4 ROI 5
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Figure 4: Time dependence of the (a) average group refractive index and (b) normalized skin thickness during OC with 40% aqueous
glycerol solution
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250
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■"1,44-
It is possible to observe a decrease in the sample thickness over the entire volume of OCT probing after applying PEG-300, this process has the highest speed and severity at the edge of the sample. When applying a 40% glycerol solution, a decrease in the sample thickness is observed in areas of the skin remote from the edge, however, at the edge of the skin, where the OPA interacts directly with the dermis, the sample thickness, on the contrary, increases.
In Figure 5, the detected volumes of the dependence of the proportion of water and PEG-300 in the skin above the OC are found, it can be seen that in the edge samples, dehydration is more pronounced and the volume fraction of OCA in the skin than in areas remote from the edge, which can be explained by the presence of lateral diffusion from the side, where the epidermis and stratum corneum are absent and OCA is very important with the dermis. At a distance of more than 2 ± 1 mm from the sample edge, no contribution from lateral diffusion was observed. The initial volume fraction of water in the samples was 70 ±2 %.
Time, min Time, min
(a) (b)
Figure 5: Time dependence of the volume fraction of (a) PEG-300 and (b) water in the skin when it is OC with PEG-300
Figure 6 shows the obtained dependence of the volume fraction of water and 40% glycerol in the skin during OC, it can be seen that dehydration is more pronounced near the edge of the sample and the volume fraction of OCA in the skin is much higher than in areas far from the edge. At a distance of more than 1 ± 0.2 mm from the edge of the sample, the contribution of lateral diffusion was not observed.
0,8-
; 0,6-
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■ ROI 1
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A ROI 3
ROI 4
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!|t|i i
0 20 40 60 80 100 120 140 160 Time, min
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Figure 6 Time dependence of the volume fraction of (a) 40% aqueous glycerol solution and (b) water in the skin when it is OC with
40% aqueous glycerol solution
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Experimental examples are given of a relatively simple method of OCT monitoring of the volume fraction of water and OCA in the skin during its ex vivo optical clearing. The main advantage of the described approach is that it allows one to simultaneously monitor changes in the geometric and optical properties of the tissue without disturbing the natural course of the OC process and obtain reliable estimates of the rate of dehydration and diffusion and the severity of these OC mechanisms under specific conditions. This method can be useful for studying the interaction of OCA with thin sections of biological tissues, for studying the features of lateral diffusion of an agent into a biological tissue, and can also serve as the basis for a new method for analyzing transdermal drug delivery.
The reported study was funded by the grant of RFBR (#20-52-56005) and the grant under the Degree of the Government of the Russian Federation No. 220 of 09 April 2010 (Agreement No.075-15-2021-615 of 04 June 2021).
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