The study of whole organs or tissues and their cellular components and structures has been historically limited by their natural opacity, which is caused by the optical heterogeneity of the tissue components that scatter light as it traverses through the tissue, making 3D tissue imaging highly challenging. In recent years, tissue clearing techniques have received widespread attention and undergone rapid development. We recently demonstrated the synthesis of a 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer. This was achieved using antipyrine (ATP) and 2,2′-thiodiethanol (TDE) as solvents. The resulting solution rapidly embedded tissue samples with a high degree of transparency and is compatible with multiple fluorescence labeling techniques. The method exhibits significant transparency effects across a range of organs, comprising the heart, liver, spleen, lung, kidney, brain (whole and sectioned), esophagus, and small intestine. It can enable volumetric imaging of tissue up to the scale of mouse organs, decrease the duration of the clearing, and preserve emission from fluorescent proteins and dyes. To facilitate the use of this powerful tool, we have provided here a detailed step-by-step protocol that should allow any laboratory to use tissue transparency technology to achieve transparency of tissues and organs.Key features• The method is primarily used for optical tissue clearing.• The method employs a HEMA-AAm copolymer for optical tissue clearing.• This method enables both 2D and 3D fluorescence imaging.

Transmission of NIR Radiation Through Multi-layer Mouse Head at Scalp Skin Optical Clearing.

Optical scattering in biological tissues presents a major challenge for achieving deep penetration in optical coherence tomography (OCT). Recent approaches leveraging the Kramers-Kronig (KK) relations suggest that strongly absorbing dyes can reduce scattering by modulating the tissue's refractive index. In this study, we investigate the use of indocyanine green (ICG), an FDA clinically approved near-infrared dye, as an optical clearing agent (OCA) for 1300 nm OCT imaging. Due to its strong absorption near 780 nm and its binding affinity to plasma proteins and lipids, ICG presents a promising candidate for KK-based optical clearing in fatty tissues. We validated ICG's clearing potential using 1300 nm OCT on the abdominal skin of a non-pigmented mouse for in vivo imaging. Our findings indicate that ICG enhances imaging contrast and increases penetration depth, demonstrating its potential for non-invasive, contrast-enhanced 1300-OCT imaging. These findings could benefit dermatological applications of OCT by enhancing image quality to reveal greater detail in the skin.

Investigations of biological samples often require sample transparency, which is achieved by embedding the sample in a high‐refractive index (RI) medium to obtain a homogenous RI distribution in the sample, referred to as optical clearing (OC). Here, we introduce a method for designing embedding media with an increased RI by increasing molecular orbitals, which is achieved by replacing elements in molecules commonly used for OC with elements possessing a more pronounced polarisability. Briefly, we took the established embedding medium Glycerol and exchanged the OH‐groups by Thiol‐groups, resulting in an embedding medium with very similar properties, but with a higher refractive index. We describe a procedure—abbreviated PRIAMOS—to render biological samples transparent using an RI‐matching liquid, which we refer to as pH‐value and Refractive Index Adjustment by Mixing highly polarisable molecular Orbital Substances. We focus on optical clearing in three‐dimensional tissue samples whilst preserving fluorescence of fluorescent labels. The clearing procedure requires 2–3 days, yielding highly transparent samples, preserving the fluorescence of fluorescent proteins like the yellow fluorescent protein (YFP). This is demonstrated on mouse brain samples, imaged with standard confocal microscopy down to 1.6 mm depth, as well as on kidney samples. Mixtures of monothioglycerol, dithioglycerol and tributylamine achieve RI values between 1.52 and 1.57, and an acidity equivalent to pH values between 5 and 8. Our PRIAMOS approach can serve as a guideline for optimising optical clearing protocols.

Advances in improving existing noninvasive optical diagnostic methods and creating new criteria for assessing the effects of diabetes on tissues and the vascular system are highly demanded. One of the promising, noninvasive and contactless methods for visualizing the vascular system is laser speckle contrast imaging. This study studies the possibility of using magnetic resonance contrast agents in laser speckle contrast imaging and optical coherence tomography, for the first time, on rat ears, under conditions of alloxan-induced type 1 diabetes. By combining diagnostic techniques, the study investigated the ability to differentiate between healthy rat tissue and diabetic rat tissue based on dynamic and structural changes caused by local application of magnetic resonance agents. This study explores the possibility of using magnetic resonance contrast agents for laser speckle-contrast imaging and optical coherence tomography of rat ears under conditions where alloxan-induced type 1 diabetes mellitus is developing was studied for the first time. Kinetics of scattering coefficient (µs) for healthy and diabetic rats during optical clearing were evaluated. The most significant reduction is seen with the Gadovist solution, showing decreases of 18.6% and 16.1% for healthy and diabetic rats, respectively. Magnevist induced a smaller reduction in the µs, with decreases of 12.6% and 6.8%, respectively. Data on the flow rate in the vessels of the ears of laboratory rats obtained in the diabetic group demonstrate reduced indicators, relative to the healthy group of animals. These results confirm the effectiveness of magnetic resonance contrast agents as optical clearing agents for both optical coherence tomography and laser speckle contrast imaging. For the first time, the nature of the change in the contrast of the static part with a change in scattering was studied. This is an additional application for studying the efficiency of optical clearing, and contributes to the multimodal application of speckle imaging methods.

Optical clearing agents (OCAs) offer a promising approach to enhance skin transparency by reducing scattering and improving photon transmission, which is critical for non-invasive optical diagnostics such as glucose sensing and vascular imaging. However, the complex multilayered structure of skin and anatomical variability across different regions pose challenges for accurately evaluating OCA performance. In this study, we developed a multilayer Monte Carlo (MC) simulation model integrated with a depth- and time-resolved diffusion model based on Fick’s law to quantitatively assess the combined effects of OCA penetration depth and refractive index change on optical clearing. The model incorporates realistic skin parameters, including variable stratum corneum thicknesses, and was validated through in vivo experiments using glycerol and glucose at different concentrations. Both the simulation and experimental results demonstrate that increased stratum corneum thickness significantly reduces blood absorption of light and lowers the clearing efficiency of OCAs. The primary influence of stratum corneum thickness lies in requiring a greater degree of refractive index matching rather than necessitating a deeper OCA penetration depth to achieve effective optical clearing. These findings underscore the importance of considering regional skin differences when selecting OCAs and designing treatment protocols. This work provides quantitative insights into the interaction between tissue structure and optical response, supporting improved application strategies in clinical diagnostics.

Dynamic imaging is a foundational tool in biology and medicine, but it is limited by light scattering in live tissues caused by refractive index mismatches, which reduces penetration depth and resolution in biological tissues. Previous work introduced a novel approach to achieve optical transparency in live animals by utilizing strongly absorbing molecules. When these absorbing molecules, such as Tartrazine, an FDA-approved dye commonly used in foods, dissolve in water, they modify the refractive index of the aqueous medium through Kramers-Kronig relations. This modulation allows reduction of the refractive index mismatch between separate components in tissue with distinct refractive indices, thus mitigating scattering. This article reports the detailed methodology of the tissue clearing technique using a Tartrazine-based aqueous solution, applied to both ex vivo and in vivo samples. This approach reversibly renders live mouse bodies transparent in the visible spectrum, enabling clearer and deeper imaging of internal processes and structures. An advanced image processing algorithm was then used to improve the contrast and segmentation of organs of interest. Additionally, high transparency in ex vivo samples, such as chicken breast,treated with Tartrazine-based solution, is shown. This method offers a versatile and accessible approach to enhanceimaging capabilities across various modalities and paves the way for advancement in the understanding of complex biological systems.

Optical coherence tomography (OCT) is a non-invasive, three-dimensional imaging modality demonstrated to yield a wealth of high-resolution structural and functional information in a variety of biomedical imaging applications. However, there is a significant limitation on the penetration depth of the modality in most tissues due to their high optical scattering, hindering imaging of more deeply embedded targets. In ophthalmic applications, this makes transscleral imaging of targets such as suprachoroidal injection volumes, lesions, and malignancies challenging. Here, we investigate the novel application of fluorescein, a commonly applied biomedical dye, as an optical clearing agent for increasing the depth of OCT imaging in scleral tissues. The effect is characterized in ex-vivo models including the sclera of porcine and human eyes, examining its time and concentration dependence, reversibility, and potential for application in the enhancement of intrasurgical trabeculectomy image guidance. We demonstrate that fluorescein can serve not only as a biomedical fluorophore, but also as an optical clearing agent capable of significantly increasing OCT imaging depth in tissue. As a common and accessible ophthalmic agent with a long history of clinical application, we believe that this new potential use could have substantial positive impact in the enhancement of biomedical imaging in scattering tissue.

Effects of hyaluronic acid with different molecular weights and concentrations on skin optical clearing and collagen fibril structural characteristics.

Measuring the density of tartrazine (TZ) powder allowed to develop a protocol for fast preparation of aqueous solutions with a desired concentration. The stability time of these solutions decreases exponentially with the increase of TZ concentration: solutions with TZ concentrations below 25% remain stable for more than 24 h, while the solution with 60% TZ remains stable only for 35 min. To validate the developed protocol, muscle samples were immersed in the 40% TZ solution and, as expected, the tissue transparency increased smoothly and exponentially during the whole treatment of 30 min. The diffusion time of TZ in exvivo skeletal muscle was quantitatively determined with high accuracy as τ TZ = 5.39 ± 0.49 min for sample thickness of 0.5 mm. By measuring the refractive index of TZ solutions during preparation, it will be easier to prepare such solutions in a fast manner for future research on tissue optical clearing.

This study investigated the use of shea butter as an optical clearing agent (OCA) to improve optical coherence tomography (OCT) imaging for early caries detection in ex-vivo human teeth. The research aimed to determine whether shea butter could enhance the contrast and clarity of OCT images, thereby aiding in the differentiation of healthy and demineralized enamel regions.she a butter was applied to the occlusal surfaces of extracted (molar) posterior teeth, and Swept Source OCT was utilized to generate two-dimensional cross-sectional images. Quantitative changes in optical attenuation coefficients and visual contrast between images captured before and after OCA application were analyzed to evaluate the effects of shea butter on imaging quality.the application of shea butter as an OCA enhanced the contrast and clarity of OCT images, particularly in distinguishing healthy from demineralized enamel. The penetration depth of OCT increased ~ 400μm and an overall increase in contrast of 30 dB is observed in the B-scans for both sound and carious tooth, offering a more comprehensive view of microstructure. Quantitative analysis revealed significant variations in the average attenuation coefficient between carious and healthy enamel regions.shea butter, a biocompatible agent, shows promise as an effective OCA for enhancing OCT imaging in dentistry. This approach could improve the early detection of carious lesions, leading to less invasive treatment options. Enhanced OCT imaging with shea butter enables better visualization of early carious lesions, facilitating preventive care, preserving tooth structure, and reducing treatment complexity.

Optical-resolution photoacoustic microscopy (OR-PAM) is a powerful imaging technique that visualizes microvascular and tissue structures with high spatial resolution, offering valuable insights into physiological and pathological processes. However, strong optical scattering in biological tissues fundamentally limits its imaging depth. Recent studies have suggested that tartrazine, a food-grade water-soluble dye, may serve as an effective optical clearing agent (OCA), yet its strong optical absorption near 532 nm raises concerns about compatibility with green-light-based OR-PAM systems. In this study, we demonstrate that by carefully controlling the concentration of tartrazine, it can effectively function as an OCA for OR-PAM at 532 nm without compromising signal quality. In vivo experiments on mouse ear and abdominal tissue showed that tartrazine significantly enhanced microvascular visibility across all tested concentrations, with the 15% weight of solute per weight of solution providing optimal clearing performance. Additionally, we evaluated 4-aminoantipyrine as another dye-based OCA, which also improved image clarity, albeit to a lesser extent. These findings highlight the promise of absorbing dye-based OCAs like tartrazine in enhancing in vivo OR-PAM by mitigating light scattering, potentially enabling deeper and clearer photoacoustic imaging in biomedical applications.

Photothermal disinfection is an emerging and efficient therapeutic method for treating various infections. However, the therapeutic efficacy is hindered by the poor tissue penetration of light. While tissue optical clearance technology can enhance light transmission, the stratum corneum impedes the delivery of clearing agents to deeper tissues. Herein, dissolving microneedle (DMN) patches incorporating optical clearing and photothermal agents are developed for improved photothermal disinfection. Such DMN patches are obtained using sucrose as the optical clearing agent and polydopamine (PDA) nanoparticles as photothermal agents in poly(vinyl alcohol) (PVA) DMN matrix. After treatment with the composite DMN, the light transmittance of the pigskin increased by 4.75 times. The composite DMNs deliver PDAs to the subsurface of skin while improving the optical clearing effect, raising the temperature of the subsurface of pigskin to 45°C under near-infrared (NIR) light irradiation with a wavelength of 808nm. In treating a deep infection model (pigskin coverage) established on mouse skin, the composite DMNs significantly improve the wound recovery rate. This approach is expected to be a general strategy for enhancing the anti-infective efficacy of photothermal disinfection and shows great potential for improving the clinical application of various phototherapies.

Tu2050: THREE-DIMENSIONAL VISUALIZATION OF HUMAN INTESTINAL TISSUES AND PRECANCEROUS LESIONS USING OPTICAL CLEARING METHODS AND STRUCTURED LIGHT ILLUMINATION IMAGING (SIM)

Photoacoustic microscopy (PAM) offers high resolution and 100% sensitivity to optical absorption, making it promising for biomedicine. However, strong light scattering in tissues limits its imaging depth, intensity, and resolution. Optical clearing agents (OCA) can reduce light scattering. However, traditional methods often use toxic substances or damage tissue components, restricting their application in living tissues. Recently, tartrazine, a common food pigment, has been shown to significantly improve tissue optical transparency while maintaining good biosafety. However, it is unclear whether tartrazine as an absorbing molecule is suitable for use in PAM. In this study, we show that tartrazine, despite its strong light absorption, can significantly enhance the performance of PAM, when used at an appropriate concentration. Our ex vivo experiments demonstrate tartrazine solution enables PAM to achieve an optical resolution of 21 μm even through the skin. A 0.6 M tartrazine solution improves resolution by 3.5 times and the imaging intensity by 4.5 times. Finally, in vivo brain imaging of a mouse with an intact scalp reveals that tartrazine not only increases the imaging intensity by about 4 times but also allows PAM to achieve an optical resolution of brain through the scalp and skull, revealing much more details of the microvasculature in the brain.

This study evaluated the efficacy of transcutaneous photodynamic therapy using blue (428 nm) LED irradiation on Staphylococcus aureus 11 in combination with a water-soluble cationic pyridyl porphyrin and optical clearing agents (OCA) in an ex vivo model. Results showed that OCA significantly enhanced photodynamic inactivation with a 61% reduction in bacterial cell counts after 15 minutes of light exposure, comparable to direct irradiation. Optical parameter analysis revealed a decrease in scattering and absorption coefficients and an increase in light penetration depth (up to 121,6%) in OCA-treated skin samples. The results confirm that optical clearing improves the efficacy of antimicrobial photodynamic action by enhancing light penetration into deeper tissue layers, reducing the need for high laser intensities, and minimizing superficial tissue damage. This approach holds promise for the treatment of skin, mucosal and soft tissue infections in humans and animals, offering valuable insights into light-tissue interactions and optimizing photodynamic therapy while reducing the risks associated with the use of LEDs and lasers.

ABSTRACTThis study evaluated the optomechanical and structural properties of individual macroscopic layers in swine skin tissues treated with a nontoxic optical clearing agent. The clearing agent was prepared by dissolving 2,2′‐thiodiethanol in a phosphate‐buffered solution and applied for up to 6 days. Prolonged clearing increased both the total and unscattered transmittance. Peaks associated with deoxygenated hemoglobin, oxygenated hemoglobin, and water showed marked reduction with extended clearing times. Histopathological examination revealed no significant structural alterations. A clearing time of approximately 4–5 days is recommended for optimal imaging, as it minimizes changes in mechanical properties. These findings support the use of optical clearing in deep tissue imaging, particularly when preserving macroscopic mechanical properties is essential, despite the inherent limitation of sample opacity.

For optical diagnosis and therapy in medicine, tracking changes in tissue refractive index (RI), absorption, and scattering coefficient is important. These characteristics may be measured using a variety of techniques. Examples of simulation and experimentation techniques for determining optical parameters are inverse Monte Carlo (IMC) simulation, integrating sphere spectroscopy (ISS), and diffuse reflectance spectroscopy (DRS). The depth of optical measurements is limited by tissue light attenuation in all methods. Using optical clearing agents (OCAs) is a common method to increase optical depth and reduce light scattering in biological tissues. Thus, optical measurement techniques employing new (OCAs) can potentially achieve greater efficiency than traditional agents. This study aims to measure the optical properties of an unsliced mouse brain in the visible spectrum using an OCA composition to clear the tissue. The mouse brain model is useful for developing neuroimaging techniques and optical monitoring of brain activity. In this study, DRS, ISS, and IMC are used to analyze the changes in the RI, absorption, and scattering coefficients of the unsliced mouse brain. A fluorescence test was additionally conducted to evaluate the efficacy of the introduced OCA in comparison to glycerol. The findings and OCA described in this study may be beneficial in optical neurostimulation and brain disease treatments.

Optical Coherence Tomography (OCT) technology offers numerous advantages, including non-contact imaging, rapid imaging capabilities, and high resolution. Although the enhancement of optical transparency through ultrasound is limited, the application of alcohol-based optical clearing agents (OCAs) combined with CaCO3 microspheres significantly improves the optical imaging of skin tissue. Using OCT, it has been demonstrated that the imaging depth of optically cleared skin tissue increases by approximately 14% due to the inclusion of CaCO3 microspheres. Molecular simulations reveal the hydrogen bonding interactions between collagen receptors, which are linked to the strong scattering characteristics of skin tissue, and four commonly used alcohol-based OCA small-molecule ligands. These simulation results correlate with the variations in imaging depth observed in experiments with different OCAs, thereby establishing the relevance and differences among various OCAs in enhancing the optical imaging effectiveness of biological tissues.

The effects of optical clearing of implantable collagen materials were studied using optical clearing agents (OCAs) based on aqueous glucose solutions of various concentrations. By measuring the kinetics of the collimated transmission spectra, the diffusion D and permeability P coefficients of the OCAs of collagen materials were determined as D = (0.22 ± 0.05) × 10-6 to (1.41 ± 0.05) × 10-6 cm2/c and P = (0.55 ± 0.04) × 10-4 to (1.77 ± 0.07) × 10-4 cm/c. Studies with optical coherence tomography (OCT) confirmed that each of the OCAs used had an effect on the optical properties of collagen materials, and allowed us to quantify the group refractive indices of the collagen of various samples, which turned out to be in the range from nc = 1.476 to nc = 1.579.