For the first time, the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing. The glucose, glycerol and propylene glycol solutions were used as clearing agents. The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers. In this contribution, the common properties of autofluorescence variation during optical immersion clearing were determined. The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail. However, the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid. The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.

Recent studies have demonstrated that topical application of glycerol on intact skin does not affect its optical scattering properties. Investigators from our research group recently revisited the use of dimethyl sulfoxide (DMSO) as an agent with optical clearing potential. We address the use of optical clearing to enhance quantitation of subsurface fluorescence emission. We employed both in vitro and in vivo model systems to study the effect of topical DMSO application on fluorescence emission. Our in vitro experiments performed on a tissue-simulating phantom suggest that DMSO-mediated optical clearing enables enhanced characterization of subsurface fluorophores. With topical DMSO application, a marked increase in fluorescence emission was observed. After 30 min, the fluorescence signal at the DMSO-treated site was 9× greater than the contralateral saline-treated site. This ratio increased to 13× at 105 min after agent application. In summary, DMSO is an effective optical clearing agent for improved fluorescence emission quantitation and warrants further study in preclinical in vivo studies. Based on outcomes from previous clinical studies on the toxicity profile of DMSO, we postulate that clinical application of DMSO as an optical clearing agent, can be performed safely, although further study is warranted.

Laser Speckle Contrast Imaging (LSCI) plays an important role in studying blood flow, but suffers from limited penetration depth of light in turbid tissue. The strong scattering of tissue obviously reduces the image contrast which decreases the sensitivity to flow velocity. Some image processing or optical clearing methods have been proposed to lessen the deficiency, but quantitative assessment of improvement is seldom given. In this study, LSCI was applied to monitor the blood flow through a capillary embedded within various tissue phantoms at depths of 0.25, 0.45, 0.65, 0.85 and 1.05 mm, and the flow velocity in capillary was controllable from 0 to 4mm/s. Here, glycerol, a common optical clearing agent, was mixed with Intralipid at different volume ratio to make the reduced scattering coefficient of tissue phantom decrease from 13.00 to 0.50 cm^-1. The quantitative analysis demonstrates that the optical clearing method can obviously enhance the image contrast, imaging depth, and sensitivity to blood flow velocity. Comparing the Laser Speckle Contrast Analysis methods and the optical clearing method, we find that for typical turbid tissue, the sensitivity to velocity estimated by the Laser Speckle Temporal Contrast Analysis (LSTCA) is twice of that by the Laser Speckle Spatial Contrast Analysis (LSSCA); while the sensitivity to velocity estimated by using the two analysis methods has a 10-fold increase, respectively, if addition of glycerol makes the reduced scattering coefficient of tissue phantom decrease by 30%. Combining the LSTCA and the optical clearing method, the sensitivity to flow velocity will be further enhanced.

One of the major challenges in imaging biological tissues using optical techniques, such as optical coherence tomography (OCT), is the lack of light penetration due to highly turbid structures within the tissue. Optical clearing techniques enable the biological samples to be more optically homogeneous, allowing for deeper penetration of light into the tissue. This study investigates the effect of optical clearing utilizing various concentrations of glucose solution (10%, 30%, and 50%) on porcine skin. A gold-plated mirror was imaged beneath the tissue and percentage clearing was determined by monitoring the change in reflected light intensity from the mirror over time. The ratio of percentage clearing per tissue thickness for 10%, 30% and 50% glucose was determined to be 4.7 ± 1.6%mm^-1 (n = 6), 10.6 ± 2.0%mm^-1 (n = 7) and 21.8 ± 2.2%mm^-1 (n = 5), respectively. It was concluded that while higher glucose concentration has the highest optical clearing effect, a suitable concentration should be chosen for the purpose of clearing, considering the osmotic stress on the tissue sample.

Optical clearing improves the penetration depth of optical measurements in turbid tissues. Polarization imaging has been demonstrated as a potentially promising tool for detecting cancers in superficial tissues, but its limited depth of detection is a major obstacle to the effective application in clinical diagnosis. In the present paper, detection depths of two polarization imaging methods, i.e., rotating linear polarization imaging (RLPI) and degree of polarization imaging (DOPI), are examined quantitatively using both experiments and Monte Carlo simulations. The results show that the contrast curves of RLPI and DOPI are different. The characteristic depth of DOPI scales with transport mean free path length, and that of RLPI increases slightly with g. Both characteristic depths of RLPI and DOPI are on the order of transport mean free path length and the former is almost twice as large as the latter. It is expected that they should have different response to optical clearing process in tissues.

The mechanism of action of clearing agents to improve optical imaging of mouse skin during reflectance-mode confocal microscopy was tested. The dermal side of excised dorsal mouse skin was exposed for one hour to saline, glycerin, or 80% DMSO, then the clearing agent was removed and the dermis placed against a glass cover slip through which a confocal microscope measured reflectance at 488 nm wavelength. An untreated control was also measured. The axial attenuation of reflectance signal, R(z_f ) versus increasing depth of focus zf behaved as R = ρ exp(-μz_f 2G), where ρ is tissue reflectivity and μ is attenuation [cm^-1]. The factor 2G accounts for the in/out path of photons, and the numerical aperture of the lens. The ρ, μ data were mapped to values of scattering coefficient (μ_s [cm^-1]) and anisotropy of scattering (g). Images showed that glycerin significantly increased the g of dermis from about 0.7 to about 0.99, with little change in the μ_s of dermis at about 300cm^-1. DMSO and saline had only slight and inconsistent effects on g and μ_s.

Our previous studies demonstrated the ultrasound-induced skin optical clearing enhancement with topical application of optical clearing agents on in vitro porcine skin and in vivo human skin. The objective of this study was to investigate the possible mechanisms of the enhanced skin optical clearing by ultrasound medications. Optical clearing effects of ex vivo guinea pig abdomen skin topically applied with 60% glycerol or the combination of 60% glycerol and ultrasound were studied by optical coherence tomography (OCT). Microstructure of skin surface was examined by scanning electron microscopy (SEM). Ultrasound with a frequency of 1MHz and a power of 0.75W over a 3-cm probe was simultaneously applied with glycerol solution for 15min. The combination of 60% glycerol and ultrasound results in a 19% increase in OCT 1/e light penetration depth after 30min, which is much better than 60% glycerol alone. SEM images demonstrated that changes in skin microstructure due to the tight order of the lipid bilayers in the stratum corneum disrupted and the separation of keratinocytes by the application of ultrasound contribute to the ultrasound-enhanced intact skin optical clearing effects.

Tissue optical clearing by use of optical clearing agents (OCAs) has been proven to have potential to reduce the highly scattering effect of biological tissues in optical techniques. However, the difference in tissue samples could lead to unreliable results, making it difficult to quantitatively control the dose of OCAs during the course of tissue optical clearing. In this work, in order to study the effects of optical clearing, we customized tissue-like phantoms with optical properties of some biological tissue. Diffuse reflectance and total transmittance of tissue-like phantoms with different OCAs (DMSO or glycerol) and porcine skin tissues were measured. Then optical property parameters were calculated by inverse adding-doubling (IAD) algorithm. Results showed that OCAs could lead to a reduction in scattering of tissue-like phantoms as it did to porcine skin tissue in vitro. Furthermore, a series of relational expressions could be fit to quantitatively describe the relationship between the doses of OCAs and the reduction of scattering effects. Therefore, proper tissue-like phantom could facilitate optical clearing to be used in quantitative control of tissue optical properties, and further promote the application potential of optical clearing to light-based noninvasive diagnostic and therapeutic techniques.

Tissue Optical Clearing Devices (TOCDs) have been shown to increase light transmission through mechanically compressed regions of naturally turbid biological tissues. We hypothesize that zones of high compressive strain induced by TOCD pins produce localized water displacement and reversible changes in tissue optical properties. In this paper, we demonstrate a novel combined mechanical finite element model and optical Monte Carlo model which simulates TOCD pin compression of an ex vivo porcine skin sample and modified spatial photon fluence distributions within the tissue. Results of this simulation qualitatively suggest that light transmission through the skin can be significantly affected by changes in compressed tissue geometry as well as concurrent changes in tissue optical properties. The development of a comprehensive multi-domain model of TOCD application to tissues such as skin could ultimately be used as a framework for optimizing future design of TOCDs.

Near-infrared spectroscopy (NIRS) technology and Mie theory are utilized for fundamental research on radiofrequency ablation of biological tissue. Firstly, NIRS is utilized to monitor rats undergoing radiofrequency ablation surgery in real time so as to explore the relationship between reduced scattering coefficient (μ^'_s) and the degree of thermally induced tissue coagulation. Then, Mie theory is utilized to analyze the morphological structure change of biological tissue so as to explore the basic mechanism of the change of optical parameters caused by thermally induced tissue coagulation. Results show that there is a close relationship between μ^'_s and the degree of thermally induced tissue coagulation; the degree of thermal coagulation can be obtained by the value of μ^'_s; when biological tissue thermally coagulates, the average equivalent scattering particle decreases, the particle density increases, and the anisotropy factor decreases.