Ex vivo confocal Raman microspectroscopy of porcinedura matersupported by optical clearing

Abstract

The effect of tissue optical clearing (TOC) to increase the probing depth and observe in‐depth structure of the ex vivo porcine dura mater was studied by confocal Raman microspectroscopy (CRM). Raman intensities were significantly increased at the depth of 250 μm for all collagen bands after treatment with glycerol. The influence of glycerol on collagen hydration was also investigated. The results indicate that the process of TOC can be divided into three main steps. The first one is a fast process of tissue dehydration accompanied by collagen shrinkage while the second relatively slow process is related to the glycerol penetration into the interfibrillar space of collagen combined with swelling of tissue. The third step is collagen dissociation caused by the high concentration of glycerol. To the best of our knowledge, this study is the first example to introduce the TOC technique in assisting CRM of ex vivo dura mater in‐depth probing.

Confocal Raman Micro-Spectroscopy for Discrimination of Glycerol Diffusivity in Ex Vivo Porcine Dura Mater

“…The DM is a connective tissue with tough and dense collagen fibrils architecture [ 66 ], which constitutes more than 90% of DM thickness. Typical CRM probing depth for DM without OC is from 175 to 250 µm, as shown in our previous study [ 44 ]. The DM collagen fibrils’ average diameter evaluated using electron microscopy is approx.…”

“…This causes reduction of delivered laser power and distorts the focused laser, which leads to a decrease the collection of Raman signal by detectors. Therefore, the Raman signal intensities become weaker with increasing depth However, the Raman band intensities for DM treated with glycerol were found to increase in the deeper layers because glycerol improved the light propagation through the upper DM layers [ 44 ], thus permitting the studying of the deeper layers.…”

“…The OC method is based on the administration of different optical clearing agents (OCAs) onto the biological objects, which reduces the scattering and absorption coefficients and thus enhances the imaging depth [ 8 , 40 , 41 , 42 ]. At present, various optical biomedical imaging methods, such as Raman spectroscopy [ 43 ], CRM [ 44 ], optical coherence tomography [ 45 , 46 ], 3D confocal microscopy [ 47 ], and polarized microscopy [ 48 ], are widely employed with various OCAs such as glucose [ 49 ], dimethyl sulfoxide (DMSO), glycerol [ 50 ], uDISCO [ 51 ], ScaleS [ 52 ], and Scale [ 53 ] to improve sensitivity in and increase light-photons-limited penetration into the biological objects.…”

“…These processes offer an additional impact on the refractive index matching with OCAs due to the water content decrease in the interstitial space. The application of OCAs can significantly affect the water concentration in the biological tissue [ 44 , 56 ]. As the hydration and water mobility states in the human skin are of great interest in cosmetology and dermatology [ 57 , 58 , 59 ], the investigation for highly effective, low-cost, non-destructive, and biocompatible OCAs with handy effect on the water concentration mobility of biological tissue for clinical use have been attracting considerable attention in recent years [ 60 , 61 , 62 , 63 ].…”

Monitoring Dermal Penetration and Permeation Kinetics of Topical Products; the Role of Raman Microspectroscopy

“…4), opening up new prospects for non-invasive, in situ/ in vivo monitoring of dermatological processes [8]. The use of optical clearing fluids has more recently been explored to reduce scattering and improve light penetration [73,74], and the state of the art of CRM for probing the physiological structure of the SC has recently been reviewed by Darvin et al [75].…”