SignificanceDiabetes can lead to the glycation of proteins and dysfunction of skin collagen. Skin lesions are a prevalent clinical symptom of diabetes mellitus (DM). Early diagnosis and assessing the efficacy of treatment for DM are crucial for patient health management. However, performing a non-invasive skin assessment in the early stages of DM is challenging.AimBy using the polarization-sensitive optical coherent tomography (PS-OCT) imaging technique, it is possible to noninvasively assess the skin changes caused by diabetes.ApproachThe PS-OCT was used to monitor the polarization characteristics of mouse skin at different stages of diabetes.ResultsBased on a multi-layered adhesive tape model, we found that the polarization characteristics (retardation, optic axis, and polarization uniformity) were sensitive to the microstructure changes in the samples. Through this method, we observed significant changes in the polarization states of the skin as diabetes progressed. This was in line with the detected microstructure changes in skin collagen fibers using scanning electron microscopy.ConclusionsThis study presents a highly useful approach for non-invasive skin assessment of diabetes.
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.
In vivo monitoring cerebral and cutaneous hemodynamics is of great important to investigate brain and peripheral circulation system functional responses to physiopathologic stimulations. Nevertheless, the high scattering characteristics of skull and skin severely limit optical imaging performance. Fortunately, in vivo tissue optical clearing techniques can efficiently overcome these problem. In this work, we combined hyperspectral imaging (HSI) and laser speckle contrast imaging (LSCI) to simultaneously monitor the changes in cortical and cutaneous microvascular blood oxygen saturation and blood flow under assistance of in vivo skull and skin optical clearing techniques, and quantitatively compared the difference between cerebral and cutaneous arteriovenous functional responses when the hypoxic stimulations was performed. The results show that the cerebral arteriovenous blood flow response is much more sensitive to the hypoxic stimulations comparing with that of cutaneous vessels. As for the arteriovenous blood oxygen response, there are only small differences between cerebral and cutaneous vessels to the instant hypoxia, but the blood oxygen level of cerebral vessels recovers faster than that of cutaneous vessels after the hypoxic stimulation. This work provides a feasible solution to realize visualization of in vivo monitoring cerebral and cutaneous microvascular reactivity with minimal invasiveness. Monitoring of microvascular reactivity with high resolution is of great significance to the study of vascular dysfunction in some peripheral vascular and cerebrovascular diseases.
Three-dimensional mapping of skeletal muscle is particularly valuable for systematical identification and analysis of various biological components, such as nerve fibers, muscle fibers and vessels during disease and regeneration. iDISCO, as a whole-mount immunolabeling technique, provides an important tool for volume imaging of muscles, but its application has been limited to thin and small muscles of young mice owing to poor antibody penetration. In this work, we developed a modified iDISCO method and applied it to label the nerve fibers in various skeletal muscles of adult mice, including diaphragm, biceps brachii and tibialis anterior. Light-sheet microscopy was used to image biceps brachii and tibialis anterior. The results showed that the modified method could achieve uniform and complete labeling of nerve fibers within muscles, whereas original iDISCO method caused strong nonspecific signal in the surface of the muscles, and intramuscular nerve fibers were almost invisible. The modified method permitted us to trace three-dimensional nerve fibers in the muscles. This method shows potential for three-dimensional histological analysis in mouse skeletal muscle, facilitating the understanding of structural-functional relationship of skeletal muscle in physiological and pathological condition.
Tissue optical clearing technique plays an important role in three-dimensional (3D) visualization of intact organs or rodent bodies. As a typical organic-solvent based clearing method, 3DISCO renders the turbid tissues transparent by the strategy of minimizing light scattering, and possesses the advantages of high transparency and substantial size reduction, which could facilitate imaging of large-volume tissues. However, for heme-rich tissues, such as spleen, liver, embryo and clinical biopsy samples, tissue transparency and image quality by 3DISCO are limited due to the strong absorption ability of hemes. To address this problem, we proposed an optimized clearing method by introducing decolorization to modify the original 3DISCO protocol. The results showed that the optimized protocol could enhance the transparency of the heme-rich tissues and facilitate high-resolution imaging inside these tissues combining with optical microscopies.
The skin is the largest organ in the human body, and it is an important window for optical imaging and diagnosis. However, the turbid characteristics of skin limits the penetration of light. Skin optical clearing technique provides an important tool for improving optical imaging performance. However, study on mechanism for skin optical clearing was mostly based on ex vivo skin experiments, the in vivo skin optical clearing process still needs further exploration. In this work, we can in vivo monitor the changes in skin during treatment of skin optical clearing agent by using two-photon imaging. For untreated skin, collagen has a strong second harmonic generation (SHG) signal, the structure of epidermis and collagen is relatively complete. After treatment of skin optical clearing agent, the SHG signal of collagen is significantly weakened. After optical clearing agent removed and saline treated, the skin can recover to initial state obviously, and the SHG is greatly enhanced, even is stronger than initial level. The results indicated that the treatment of optical clearing agents (OCA) can induce the structural change of skin, which lead to skin transparent consequently, and optical clearing agent–induced decrease of collagen SHG is invertible by recovery with treatment of physiological saline. This work is very helpful for understanding the in vivo skin optical clearing process, which is of great significant to revealing the mechanism of in vivo skin optical clearing.
To monitor skin microvascular dysfunction of alloxan-induced type 1 diabetic mice model. In this work, we used laser speckle contrast imaging and hyperspectral imaging through in vivo skin optical clearing method to simultaneously monitor the noradrenaline-induced response of microvascular blood flow and blood oxygen with the development of diabetes. The main results showed that venous and arterious blood flow steadily decreased without recovery after injecting noradrenaline (NE), furthermore the influence of NE-induced arterious blood oxygen response greatly decreased, especially for 2-weeks and 4-weeks diabetic mice. This study demonstrated that skin microvascular function was a potential research biomarker for early warning in the occurrence and development of diabetes. And it provides a feasible solution to realize visualization of cutaneous microvessels for monitoring microvascular reactivity.
Skin optical clearing can significantly enhance the ability of biomedical optical imaging. Some alcohols and sugars have been selected to be optical clearing agents (OCAs). In this work, we paid attention to the optical clearing potential of disaccharides. Sucrose and maltose were chosen as typical disaccharides to compare with fructose, an excellent monosaccharide-OCA, by using molecular dynamics simulation and an ex vivo experiment. The experimental results indicated that the optical clearing efficacy of skin increases linearly with the concentration for each OCA. Both the theoretical predication and experimental results revealed that the two disaccharides exerted a better optical clearing potential than fructose at the same concentration, and sucrose is optimal. Since maltose has an extremely low saturation concentration, the other two OCAs with saturation concentrations were treated topically on rat skin in vivo, and optical coherence tomography imaging was applied to monitor the optical clearing process. The results demonstrated that sucrose could cause a more significant increase in imaging depth and signal intensity than fructose.
The developed optical clearing methods show great potential for imaging of large-volume tissues, but these methods present some nonnegligible limitations such as complexity of implementation and long incubation times. In this study, we tried to screen out rapid optical clearing agents by means of molecular dynamical simulation and experimental demonstration. According to the optical clearing potential of sugar and sugar-alcohol, we further evaluated the improvement in the optical clearing efficacy of mouse brain samples, imaging depth, fluorescence preservation, and linear deformation. The results showed that drops of sorbitol, sucrose, and fructose could quickly make the mouse brain sample transparent within 1 to 2 min, and induce about threefold enhancement in imaging depth. The former two could evidently enhance the fluorescence intensity of green fluorescent protein (GFP) and prodium iodide (PI) nuclear dye. Fructose could significantly increase the fluorescence intensity of PI, but slightly decrease the fluorescence intensity of GFP. Even though the three agents caused some shrinkage in samples, the contraction in horizontal and longitudinal directions are almost the same.
The improvement of methods for optical clearing agent prediction exerts an important impact on tissue optical clearing technique. The molecular dynamic simulation is one of the most convincing and simplest approaches to predict the optical clearing potential of agents by analyzing the hydrogen bonds, hydrogen bridges and hydrogen bridges type forming between agents and collagen. However, the above analysis methods still suffer from some problem such as analysis of cyclic molecule by reason of molecular conformation. In this study, a molecular effective coverage surface area based on the molecular dynamic simulation was proposed to predict the potential of optical clearing agents. Several typical cyclic molecules, fructose, glucose and chain molecules, sorbitol, xylitol were analyzed by calculating their molecular effective coverage surface area, hydrogen bonds, hydrogen bridges and hydrogen bridges type, respectively. In order to verify this analysis methods, invitro skin samples optical clearing efficacy were measured after 25 min immersing in the solutions, fructose, glucose, sorbitol and xylitol at concentration of 3.5 M using 1951 USAF resolution test target. The experimental results show accordance with prediction of molecular effective coverage surface area. Further to compare molecular effective coverage surface area with other parameters, it can show that molecular effective coverage surface area has a better performance in predicting OCP of agents.
Molecular dynamics (MD) simulation provides a quantitative method for screening efficient optical clearing agents. Some excellent optical clearing agents were obtained through the MD simulation of alcohols with hydroxyl groups. Among of the agents, fructose, one of polyols has the better efficacy which is one of monoaccharides. In comparison, disaccharides received little attention. In this study, sucrose and maltose were screened from common disaccharides for comparing with fructose. Actually, each agent has different saturation concentration. Here, the combination of molecular dynamic simulation and in vitro experiment was employed in this study. Firstly, the hydrogen bonds were used to predict the optical clearing potential of sucrose, maltose and fructose by using molecular dynamic simulation, respectively. The simulated results indicated that the optical clearing potential in a descending order as: sucrose, maltose and fructose. The reduced scattering coefficient of in vitro rat skin samples was used to evaluate the skin optical clearing potential based on measurements of reflectance and transmittance using a commercial spectrophotometer with an integrating sphere. Both the experimental and simulated results show that for the same concentration, disaccharides have better optical clearing potential than fructose. However, maltose has too low saturation concentration to produce similar optical clearing efficacy as the other two. By contrast, sucrose is the best one even though it has lower saturation concentrations than fructose.
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