Optical clearing of tissues and organs based on the kramers-kronig relation

Abstract

In one aspect, the disclosure relates to a method for imaging an organ or tissue in a subject, the method including at least the step of administering a composition comprising a clearing compound to the subject, wherein an interaction between the clearing compound and at least one overlying tissue in the subject creates a transparent spectral window in the at least one overlying tissue. In some aspects, the transparent spectral window can be the entire visible spectrum or a portion thereof. In another aspect, the clearing compounds are non-toxic and are excreted by the subject following imaging. Also disclosed herein are pharmaceutical compositions including the clearing compounds and kits including the pharmaceutical compositions. In any of these aspects, the subject can be a living subject; the method can also be performed in an ex vivo tissue or organ.

Description

ATTORNEY DOCKET NO.221910-2720 OPTICAL CLEARING OF TISSUES AND ORGANS BASED ON THE KRAMERS-KRONIG RELATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 690,096, filed September 5, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No.2045120 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND

[0003] Effective tissue clearing is vital for enabling deep, high-resolution imaging of internal biological structures, directly impacting the precision of diagnostics, therapeutic interventions, and advanced imaging techniques. Traditional clearing methods, including hydrophobic, hydrophilic, and hydrogel-based approaches, can effectively reduce scattering but are unsuitable for live tissues due to their toxicity or disruption of essential biological components, thus precluding their use in live organisms. Thus, they are incompatible with real-time, in vivo imaging of dynamic physiological processes.

[0004] Biomedical imaging plays a central role in clinical analysis and medical intervention while allowing for non-invasive studies of complex biological processes. However, optical imaging of biological tissues is fundamentally limited by scattering and absorption of light. In most tissues, the scattering coefficient is 10−1000 times larger than the absorption coefficient; thus, scattering processes can severely limit the imaging depth and spatial resolution in conventional microscopy. For this reason, the ability to achieve significant reductions in light scattering holds promise for transforming brightfield, fluorescence, nonlinear, and super-resolution imaging techniques.

[0005] Light scattering in tissue originates from the optical contrast between low refractive index (RI) aqueous-based components (e.g., the interstitial fluid and cytosol) and high RI lipid-based components (e.g., the plasma membrane, myelin, and myofibrils). Existing methods to reduce optical contrast usually replace water with high-RI chemicals or remove lipids to yield an all- aqueous environment. Despite their success, these approaches are seldom employed in live tissues as they involve the use of toxic substances (e.g., tetrahydrofuran and acrylamide) and removal of molecules vital to sustaining life (e.g., water and lipids).ATTORNEY DOCKET NO.221910-2720

[0006] Major advances in medicine and biology have been driven by imaging techniques such as microscopy, X-ray, and Magnetic Resonance Imaging (MRI). While methods like MRI and X-ray provide indirect measurements of tissue function, they are limited in capturing changes at biological timescales. Current non-invasive optical imaging techniques offer rapid assessment of tissue function and structure but are hindered by strong light scattering in biological tissues, restricting penetration depth and resolution.

[0007] Previous approaches to achieving optical transparency in vivo rely on dye molecules with major absorption peaks in the visible spectrum, such as tartrazine, which features peak absorption at 428 nm and lingering absorption up to 600 nm (FIG.5A). Consequently, optical transparency is limited to wavelengths above 600 nm, restricting the application of this approach for imaging shorter-wavelength fluorophores, such as commonly used green and yellow fluorescent proteins (GFP and YFP, respectively).

[0008] It would be desirable to develop non-invasive optical imaging techniques for the purpose of assessing tissue function and structure through the use of clearing compounds and / or compositions that reduce light scattering in biological tissues. It would further be desirable if use of the clearing compounds enabled greater penetration depth and resolution than current methods. It would further be desirable if compounds and / or compositions enabling the clearing were nontoxic, biocompatible, and inexpensive, and allowed tissue transparency across the entire visual spectrum. These needs and other needs are satisfied by the present disclosure. SUMMARY

[0009] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to a method for imaging an organ or tissue in a subject, the method including at least the step of administering a composition comprising a clearing compound to the subject, wherein an interaction between the clearing compound and at least one overlying tissue in the subject creates a transparent spectral window in the at least one overlying tissue. In some aspects, the transparent spectral window can be the entire visible spectrum or a portion thereof. In another aspect, the clearing compounds are non-toxic and are excreted by the subject following imaging. Also disclosed herein are pharmaceutical compositions including the clearing compounds and kits including the pharmaceutical compositions. In any of these aspects, the subject can be a living subject; the method can also be performed in an ex vivo tissue or organ.

[0010] Other systems, methods, features, and advantages of the present disclosure will be orATTORNEY DOCKET NO.221910-2720 become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0012] FIGs. 1A-1N show the physical mechanism for achieving optical transparency with absorbing molecules. (FIG.1A) Numerical simulation showing the modulation of the real index of an aqueous solution (nʹ, green solid line) by introducing an absorbing molecule with a peak in nʹʹ (gray dashed line) centered at 428 nm. The nʹ of water (blue solid line) and high-RI cellular components (e.g., lipids and collagen fibers) are also shown. (FIGs.1B-1C) Imaginary part nʹʹ (FIG.1B) and change in the real part Δnʹ (FIG.1C) of the RI calculated from a Lorentz oscillator model with resonance frequency ω0 at 100 nm (blue), 250 nm (green) and 400 nm (red), respectively. (FIGs. 1D-1E) Molar absorption α (FIG. 1D) and molar n' change β (FIG. 1E) of glycerol (blue), antipyrine (green), and tartrazine (red) dissolved in water, respectively. The data for glycerol below 250 nm was obtained from the literature. (FIG.1F) Imaginary part of refractive index, n'', of tartrazine solutions at different concentrations measured by ellipsometry. (FIG.1G) Dependence of n'' on molar concentrations at 430 nm. Dashed lines represent linear fitting of the data, from which molar absorption α is extracted. (FIG.1H) Real part of refractive index, n', of tartrazine solutions at different concentrations measured by ellipsometry. (FIG.1I) Dependence of n' on molar concentrations at 500 nm, 600 nm, 700 nm, and 800 nm. Dashed lines represent linear fitting of each curve, from which the slopes are extracted to represent the molar n' change, β, at each wavelength. (FIG.1J) Transmittance T of tartrazine solutions with an optical path length of 1 mm measured at different concentrations, showing the transmission window beyond 600 nmATTORNEY DOCKET NO.221910-2720 despite strong absorption below 500 nm. (FIGs.1K-1L) Wavelength dependence of maximum molar absorption α (K) and maximum molar n' change β (FIG.1L) for glycerol, antipyrine, and 21 absorbing molecules listed in Table 2. (FIG.1M) The relation between average molar absorption α and average molar n' change β in the visible spectrum for different dye molecules. Tartrazine is highlighted as Dye-4 in the red dotted circle. Dye-21 is not shown due to the large negative β in the wavelength range of interest. (FIG.1N) The ratio between the molar n' change and molar absorption (β / α) for different absorbing molecules. The “white window” indicates a potential transparent spectrum with sufficient ∆n' and minimal absorption.

[0013] FIGs.2A-2J show high-resolution dynamic imaging of the mouse enteric nervous system through a transparent abdomen. (FIG.2A) Schematics showing microscopic imaging through the transparent abdomen of mice. (FIGs. 2B-2C) Fluorescence images of the mouse myenteric plexus before (FIG.2B) and after (FIG.2C) optical transparency is achieved. (FIG.2D) Widefield fluorescence image series of the mouse myenteric plexus, overlaid with the illustration of local displacement mapping. The direction and magnitude of movement are represented in corresponding colors and arrows. (FIG.2E) Temporal evolution of the average moving direction and displacement magnitude over intervals of 0.36 s in a 220✕220 μm region. (FIG.2F) Spatial- temporal evolution of plexus displacement between consecutive frames with direction encoded in the background color and magnitude in vector lengths. (FIG. 2G) A snapshot highlighting the diverse patterns of local plexus movements in a representative frame. Dashed boxes highlight contraction (blue), expansion (red), and rotation (orange), respectively. (FIGs. 2H-2J) Three representative local movement patterns observed: contraction (FIG.2H), expansion (FIG.2I), and rotation (FIG.2J). The colormap denotes divergence in FIGs.2H-2I and curl in FIG.2J, calculated from the displacement field, respectively. All scale bars denote 50 μm.

[0014] FIGs.3A-3F show generalizability of the Lorentz model in guiding the selection of dye molecules for achieving in vivo optical transparency. (FIGs. 3A-3B) Laser speckle contrast images of the mouse head before (FIG.3A) and after (FIG.3B) topical application of tartrazine. (FIGs.3C-3D) Laser speckle contrast images of the mouse head before (FIG.3C) and after (FIG. 3D) topical application of 4-aminoantipyrine, a red-shifted version of antipyrine. (FIGs. 3E-3F) Laser speckle contrast images of the mouse head before (FIG.3E) and after (FIG.3F) topical application of sunset yellow, another common food pigment with yellow-to-orange color. Scale bars: 5 mm.ATTORNEY DOCKET NO.221910-2720

[0015] FIGs.4A-4E show n' and n'' spectra of several representative dyes in Table 2. Real part (n', left axis, solid lines) and imaginary part (n'', right axis, dashed lines) of the RI of (FIG.4A) Dye-5 at 1 M, (FIG.4B) Dye-7 at 1 M, (FIG.4C) Dye-17 at 0.3 M, and (FIG.4D) Dye-16 at 0.5 M. (FIG.4E) The transmission spectra of different dyes measured through a 1-mm path length.

[0016] FIGs.5A-5G show optical characterizations of ampyrone. (FIG.5A) Comparison of the mass extinction coefficients of phenazone, ampyrone, and tartrazine solutions as a function of wavelength, highlighting the distinct positions of their absorption edges. A 100 mg / mL aqueous solution was used for each spectrum. (FIG. 5B) The real RI (n) of an aqueous solution of ampyrone as a function of concentration and wavelength. (FIG.5C) The real RI (n) of an aqueous solution of ampyrone as a function of concentration at selected wavelengths. (FIG. 5D) The imaginary RI (k) of an aqueous solution of ampyrone as a function of concentration and wavelength. (FIG.5E) Comparison of k for various index-matching agents in aqueous solutions at 310 nm as a function of concentration. (FIG.5F) Comparison of the real RI (n) for the same index-matching agents shown in FIG.5E as a function of concentration at 310 nm. The Dextran data had to be linearly extrapolated up to 50% w / w since high concentrations are not achievable due to its solubility limit. Data in FIGs.5C and 5E-5F are shown as mean ± standard deviation (SD) from three wavelength-dependent measurements.

[0017] FIGs. 6A-6D show resolution characterization of scattering phantoms. (FIG. 6A) Phantoms of 1 μm silica beads in 5-mm-thick scattering phantoms composed of agarose hydrogel with increasing concentrations of ampyrone. Scale bars are 5 mm. (FIG. 6B) 1951 USAF resolution test target images through 1 μm silica beads in 2-mm-thick scattering phantoms. The scale bar is 1.2 mm. (FIG.6C) Same test target images as in FIG.6B, but zoomed in to display the smallest features. The scale bar is 296 μm. (FIG.6D) Modulation transfer functions (MTFs) for all concentrations of ampyrone used in FIGs. 6B-6C with the same color coding by concentration. Each subplot shows the MTFs at different concentrations for a single wavelength.

[0018] FIGs.7A-7D show achieving optical transparency in ex vivo mouse skin. (FIG.7A) Mouse abdominal skin before (top) and after (bottom) soaking in ampyrone solutions at various concentrations. (FIG.7B) Transmission spectra through the same four mouse skin samples as in FIG.7A after soaking. (FIG.7C) The ratio of the transmission after soaking to before soaking for the same four mouse skin samples. (FIG.7D) The area change after soaking for the same four skin samples is shown as a function of concentration. All scale bars are 5 mm.ATTORNEY DOCKET NO.221910-2720

[0019] FIGs.8A-8D show achieving optical transparency in the live mouse abdomen. (FIG.8A) Mouse abdomen before treatment with ampyrone solution. (FIG. 8B) Mouse abdomen shown after treatment with ampyrone solution. (FIG.8C) The same mouse abdomen in FIGs.8A-8B after dissection. Scale bars are 1 cm in FIGs.8A-8C. (FIG.8D) Images showing the abdominal area of the same mouse before treatment, after achieving a transparent window with ampyrone, after reversing the transparency effect, and hair regrowth on the subsequent days. Scale bar is 1 cm in FIG.8D.

[0020] FIGs.9A-9N show longitudinal neuron structural imaging through transparent scalp. (FIG. 9A) 3D reconstruction of two-photon excited YFP-H fluorescence in the live mouse cortex before treatment with ampyrone. Only YFP signals in the scalp can be seen due to the scattering of the scalp. (FIG.9B) 3D reconstruction of two-photon excited YFP-H fluorescence of the same region as FIG.9A after achieving scalp transparency with ampyrone. The scalp, skull, and brain (to the imageable depth) are labeled in the figure. Their approximate thicknesses are 150^μm for the scalp, 100^μm for the skull, and 400^μm for the imageable depth of the brain, respectively. (FIGs. 9C-9D) Images of the same brain region before treatment with ampyrone at 300 μm and 400 μm beneath the cortical surface, respectively. (FIGs.9E-9F) Images of the same brain region after achieving scalp transparency with ampyrone at 300 μm and 400 μm beneath the cortical surface, respectively. (FIGs.9G-9J) 3D reconstruction of two-photon excited YFP-H fluorescence of the same region in layer 1 of the primary visual area (V1) through the transparent window in the scalp on P22-P25. Yellow and blue colors indicate YFP and SHG, respectively. (FIGs. 9K-9N) YFP images of the same region in layer 1 of V1 at a depth of 250 μm below the surface of the scalp on P22-25. The same vascular landmarks, appearing as dark linear features, are present in all images, confirming longitudinal imaging in the same mouse brain. Scale bars are 100 μm in FIGs. 9A-9F and 200 μm in FIGs.9G-9N.

[0021] FIGs.10A-10F show functional neuron activity imaging with GCaMP through transparent scalp. (FIG.10A) 3D reconstruction of two-photon excited GCaMP8m fluorescence in the mouse cortex before treatment with ampyrone. Only GCaMP8m signals in the scalp can be seen due to the scattering of the scalp. (FIG.10B) 3D reconstruction of the same GCaMP8m-labeled mouse cortex after achieving transparency in the scalp. (FIG.10C) Image from the same ROI as FIG. 10A at 170 μm depth from the surface of the scalp. (FIG.10D) Image from the same ROI as FIG. 10B at 170 μm depth showing dense cell bodies. (FIG.10E) A representative time-frame image of the cortex of a GCaMP6f-labeled mouse at 200 μm depth from the surface of the scalp. Representative neurons are labeled with white circles. (FIG. 10F) Dynamic time traces ofATTORNEY DOCKET NO.221910-2720 GCaMP6f fluorescence intensity corresponding to the labeled neurons in FIG. 10E. Red lines indicate the stimulus applied to the mouse. Scale bars are 100 μm in FIGs.10A-10B, 200 μm in FIGs.10C-10D, and 400 μm in FIG.10E. The different strain, larger FOV, use of awake mice, and lower laser power used for functional imaging in FIG. 10E are responsible for the lower resolution of cell bodies in FIG.10E compared to FIG.10D.

[0022] FIGs.11A-11C show proteomics results of mouse abdominal skin. (FIG.11A) Schematic of protein extraction from mouse abdominal skin following a 72 h recovery period post treatment with ampyrone. (FIGs.11B-11C) Volcano plot depicting the female (FIG.11B) and male (FIG. 11C) mouse proteomics results with potential apoptotic markers highlighted. Dashed, horizontal lines correspond to the 0.05 P-value threshold for significance and vertical lines correspond to fold changes of ±2, which represent Log2(2+1). The fold change is defined as (final value – original value) / (original value).

[0023] FIGs. 12A-12D show Kramers-Kronig simulations of absorption spectra modeled as Gaussian and boxcar functions. (FIG.12A) Absorption spectra modeled as Gaussian functions (blue) with the corresponding RI modulation (orange). The standard deviation of the Gaussian function, σ, is set to 10, 50, and 100 nm with the height kept constant. (FIG.12B) Absorption spectra modeled as Gaussian functions (blue) with the corresponding RI modulation (orange). The standard deviation of the Gaussian function, σ, is set to 10, 50, and 100 nm with the area under the curve kept constant. (FIG.12C) Absorption spectra modeled as boxcar functions (blue) with the corresponding RI modulation (orange). The width of the boxcar function is set to 20, 100, and 200 nm with the height kept constant. (FIG.12D) Absorption spectra modeled as Gaussian and boxcar functions with the same area under the curve (blue) alongside the corresponding RI modulation (orange). These plots, taken together, show that far-off-resonance RI modulation depends on total area under the curve of the absorption spectrum, while near-resonance depends on the sharpness of the absorption peak.

[0024] FIGs. 13A-13E show ampyrone comparison under different storage conditions. The “Ampyrone, Air” sample was stored at room temperature in air in a dark cabinet for over 1 year. The “Ampyron, N2” sample was stored in a nitrogen purged glove box at -20 °C in darkness for approximately 3 months. (FIG. 13A) UV-Vis spectra of ampyrone under nitrogen and air at a concentration 0.1 mg / mL. (FIG.13B) Visual comparison of both samples at a concentration of 100 mg / mL. (FIG.13C) UV-VIS spectra of ampyrone at 100 mg / mL to demonstrate absorption differences around 400 nm between the two samples. (FIG.13D) Real part of the refractive indexATTORNEY DOCKET NO.221910-2720 comparison for both samples at 100 mg / mL. (FIG. 13E)1H NMR spectra of both ampyrone samples at 100 mg / mL in D2O.

[0025] FIGs.14A-14H show ellipsometry data for all compounds in FIGs.5A-5G. Real RI spectra of (FIG. 14A) phenazone, (FIG. 14B) sucrose, (FIG. 14C) glycerol, and (FIG. 14D) dextran. Imaginary RI spectra of (FIG.14E) phenazone, (FIG.14F) sucrose, (FIG.14G) glycerol, and (FIG. 14H) dextran. Black shading in the plots (FIGs.14D and 14H) indicates unavailable data due to solubility constraints.

[0026] FIGs.15A-15P show blood panel results. Mice in the experimental group were topically treated with 38% w / w ampyrone (red) while mice in the control group were treated with 1x PBS in an identical manner (black). All mice were allowed to recover using a topically applied hydrogel. D1 mice were collected after 1 day of recovery from topical application of ampyrone or PBS. D14 mice were collected after 14 days of recovery. (FIG.15A) White blood cells. (FIG.15B) Red blood cells. (FIG.15C) Hemoglobin. (FIG.15D) Hematocrit. (FIG.15E) Mean corpuscular volume. (FIG. 15F) Mean corpuscular hemoglobin. (FIG. 15G) Mean corpuscular hemoglobin concentration. (FIG.15H) Platelet count. (FIG.15I) Neutrophils. (FIG.15J) Lymphocytes. (FIG.15K) Monocytes. (FIG. 15L) Eosinophils. (FIG. 15M) Glucose. (FIG. 15N) Aspartate transaminase. (FIG. 15O) Alanine transaminase. (FIG.15P) Alkaline phosphatase. Sample size is n=8 mice for PBS D1, n=7 for ampyrone D1, n=7 for PBS D14, and n=8 for ampyrone D14. Bar graphs are shown as mean values ± SD.

[0027] FIGs. 16A-16H show histology results for mice abdominal skin treated with PBS and ampyrone. (FIGs.16A-16H) Histology images of abdominal skin from representative female mice on 1 day after treatment with PBS (FIG.16A), 1 day after treatment with ampyrone (FIG.16B), 14 days after treatment with PBS (FIG.16C), and 14 days after treatment with ampyrone (FIG. 16D). (FIGs.16E-16H) Histology images of abdominal skin from representative male mice on 1 day after treatment with PBS (FIG.16E), 1 day after treatment with ampyrone (FIG.16F), 14 days after treatment with PBS (FIG.16G), and 14 days after treatment with ampyrone (FIG.16H). For all day 1 images, arrows indicate ulceration and asterisks indicate dermal necrosis while for all day 14 images, arrows indicate dermal fibrosis and asterisks indicate follicular and adnexal drop out. Scale bars are 100 μm in FIGs.16A-16B and 16E-16F and 200 μm in FIGs.16C-16D and 16G-16H.ATTORNEY DOCKET NO.221910-2720

[0028] FIGs. 17-17B show hair follicle images from untreated mouse scalp. (FIG. 17A) 3D visualization with a scale bar representing 100 μm. (FIG.17B) 2D image showing the distribution of hair follicles with a scale bar of 100 μm.

[0029] FIGs.18A-18E show the origin of line-like structures in images through transparent scalp. (FIGs. 18A-18D) Two-photon excited fluorescence images of YFP-H-labeled cortex from the same live mouse through untreated scalp (FIG. 18A), through treated transparent scalp (FIG. 18B), after scalp removed with the skull intact (FIG.18C), and after removal of both scalp and skull via a cranial window (FIG. 18D). Scale bars in A-D: 100 μm. (FIG. 18E) FWHM measurements of the smallest resolvable line-like structures under each condition. All data are reported as mean ± standard deviation, SD, with data points shown for n = 10 line features in each group. NS: not significant (p>0.05).

[0030] FIGs.19A-19D show lateral resolution measurements with fluorescent beads embedded in agar. (FIG. 19A) Full width at half maximum (FWHM) measurements of 200-nm-diameter fluorescent beads in agar underneath the ampyrone-treated transparent scalp and skull. (FIG. 19B) FWHM measurements of the fluorescent beads underneath the skull only. (FIG. 19C) FWHM measurements of the fluorescent beads in agar without any overlying tissue. The solid and dashed lines in FIGs.19A-19C represent the mean ± standard deviation (SD) from three independent measurements. (FIG.19D) Measurements were taken of two-photon fluorescence intensity under excitation laser transmitted through: the cleared scalp and skull, the skull with the scalp removed, and neither scalp nor skull. All intensity values are normalized to the condition without scalp or skull. A 6x power attenuation was observed for the skull, attributed to persistent scattering within the bone, as ampyrone cannot penetrate hard bone tissue. Moreover, an additional 4x power attenuation was found for the cleared scalp, primarily due to absorption by pigments in the hair follicles, the presence of which is confirmed by FIGs.17A-17B. However, when the scalp is uncleared, no fluorescence signal can be detected from the brain through the uncleared scalp and skull due to the strong scattering in the scalp. Data in FIG.19D are shown as mean ± standard deviation (SD) from three independent measurements.

[0031] FIGs. 20A-20I show individual neuron traces from FIGs. 10A-10F. (FIGs. 20A-20F) Individual traces of the dynamic calcium signals corresponding to those in FIG.10F. Red lines indicate the stimulus applied to the mouse. (FIG.20G) Average (black) of all transients for neuron 1 as an example of a responsive neuron. Individual traces are shown in gray. (FIG.20H) Average (black) of all transients for neuron 4 as an example of a non-responsive neuron. Individual tracesATTORNEY DOCKET NO.221910-2720 are shown in gray. (FIG.20I) Peak responses for all 6 neurons, with the red data for the active neurons showing a much greater peak response than their inactive counterparts. The P-values between the peak and baseline responses for neurons 1-3 are 0.00055 (***), 0.019 (*), and 0.0022 (**). The P-values between the peak and baseline responses for neurons 4-6 are 0.058, 0.066, and 0.92 (NS, not significant).

[0032] FIGs.21A-21I show a comparison of GCaMP6f images of the same cortical region in a mouse through the opaque scalp, through the transparent scalp, and with the scalp removed. (FIG.21A) GCaMP6f images of the mouse cortex through the untreated, opaque scalp, showing no discernible features. (FIG.21B) GCaMP6f image of the mouse cortex through the transparent scalp and intact skull. (FIG.21C) GCaMP6f image of the mouse cortex with scalp removed and intact skull. (FIG.21D) The power spectral density (PSD) map of the image in FIG.21A. (FIG. 21E) The PSD map of the image in FIG.21B. (FIG.21F) The PSD map of the image in FIG.21C. In the PSD maps, low spatial frequencies are concentrated at the center, while higher spatial frequencies increase toward the edges. (FIGs. 21G-21I) Normalized radial PSD of the mouse cortex through untreated scalp (FIG.21G), treated scalp (FIG.21H), and bare skull (FIG.21I). The normalized radial PSD results suggest that the resolvable higher spatial frequencies are comparable when imaging through the transparent scalp and without the scalp, but are absent when imaging through the untreated scalp. Scale bars are 200 μm in FIGs.21A-21C.

[0033] FIGs.22A-22D show two-photon imaging setups for live mice. (FIG.22A) Two-photon fluorescence microscopy setup for a mouse under anesthesia, showing the heating pad (red arrow) that maintains the mouse body temperature during imaging. (FIG.22B) The same two- photon fluorescence microscopy setup as in FIG. 22A at higher magnification, showing the coverslip holder (green arrow) that isolates the objective from the mouse. (FIG.22C) Two-photon fluorescence microscopy setup for an awake mouse, showing the treadmill (yellow arrow) used to minimize movement and reduce stress. (FIG. 22D) The same two-photon fluorescence microscopy setup as in FIG.22C, showing an air tube (blue arrow) used to deliver an air-puff stimulus to the mouse whiskers and face during calcium imaging. The anesthetized setup was used for structural, non-longitudinal YFP imaging and for GCaMP imaging with a 20X immersion objective, while the awake setup was used for longitudinal YFP imaging and functional GCaMP imaging with a 10X air objective. For scale, the diameter of the optical posts in each of the images is 12.7 mm.ATTORNEY DOCKET NO.221910-2720

[0034] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. DETAILED DESCRIPTION

[0035] The complex structure of biological matter causes opacity due to unwanted scattering and absorption of light, which limits the penetration depth of optical imaging. In most tissues, the scattering coefficient is 10−1000 times larger than the absorption coefficient; thus, scattering processes can severely limit the imaging depth and spatial resolution in conventional microscopy. If one could achieve significant reductions in light scattering, this could lead to significant enhancement of brightfield, fluorescence, nonlinear, and super-resolution imaging techniques.

[0036] Light scattering in tissue originates from the difference between low refractive index (RI) aqueous-based components (e.g., the interstitial fluid and cytosol) and high RI lipid- and protein- based components (e.g., the plasma membrane, myelin, and myofibrils). Existing methods to reduce this difference in RI usually replace water with high-RI chemicals or remove lipids to yield an all-aqueous environment. Despite their success, these approaches are seldom employed in live tissues as they involve the use of toxic substances (e.g., tetrahydrofuran and acrylamide) and removal of molecules vital to sustaining life (e.g., water and lipids).

[0037] Tissue-clearing techniques enhance the optical transparency of biological samples by minimizing refractive index (RI) differences between tissue components, thereby reducing light scattering. This reduction in scattering allows light to penetrate deeper into tissues, enabling high- resolution imaging of large tissue volumes using light microscopy. collection of approaches has been developed to achieve transparency in biological tissue, such as Scale, which uses sorbitol and urea, and CLARITY, replacing lipids with a hydrogel matrix, have significantly advanced imaging capabilities. Other notable techniques include CUBIC, which employs amino alcohols for rapid clearing, PACT for embedding and clearing soft tissue, and ClearT, optimized for embryonic and small samples. Each of these tissue clearing technologies offers distinct advantages in speed, tissue compatibility, and fluorescence preservation, leading to widespread applications in neuroscience, oncology, and developmental biology. However, these protocols commonly require tissue fixation, remove either lipids or water to form a homogeneous RI-matched environment,ATTORNEY DOCKET NO.221910-2720 and rely on potentially toxic clearing agents (e.g., dichloromethane, benzyl alcohol). Such constraints limit their suitability for in vivo use and often demand lengthy processing times spanning several days. Moreover, most existing techniques only provide narrow spectral transparency, restricting their utility to specific imaging wavelengths.

[0038] In one aspect, by extending skin transparency across the entire visible spectrum, the disclosed method enables noninvasive imaging of both YFP and the GFP-based calcium sensor GCaMP in the live mouse brain without removing either the scalp or skull. In another aspect, the disclosed creation of a color-neutral transparency window in the mouse scalp allows repeated longitudinal imaging of the same brain regions in the same mouse throughout development. In a further aspect, this advancement opens the door to noninvasive deep-tissue optical imaging, providing long-term visualization of cellular structures and dynamic activity with high spatiotemporal resolution and chronic tracking capabilities.

[0039] In one aspect, existing methods for tissue transparency require toxic organic solvents having a high refractive index to reduce the refractive index mismatch between scatterers and aqueous background, or to remove scatterers inside biological tissues. In a further aspect, existing methods can only be applied to achieve optical transparency in fixed tissues from specific organs of interest, for example in post mortem examination or post-surgical histological examination. In some aspects, existing methods involve the replacement of original tissue components with exogenous chemicals, including, but not limited to, replacement of cellular lipids with hydrogels. In a further aspect, existing methods may further involve electrophoresing an excised specimen, exposing the specimen to hydrodynamic pressure, microwave radiation, or ultrasonic vibration.

[0040] In still another aspect, existing methods may be able to preserve three-dimensional structures of tissues, but the tissues must still be removed from the body of a subject and, for example, directly contacted with an exogenous component or composition such as, for example, a tissue clearing composition, a surfactant (e.g. a non-ionic surfactant such as a saponin), a buffer, an enzyme, an anticoagulant, a solvent (e.g. acetone), a non-ionic density gradient medium (e.g. a phthalimide), or any combination thereof. In one aspect, existing methods employing such exogenous components as listed herein still require tissue removal from the subject for achieving optical transparency. In a further aspect, the present methods do not require use of some or all of the above-listed components.ATTORNEY DOCKET NO.221910-2720

[0041] By contrast, in one aspect, the disclosed method is based on refractive index modulation of existing tissue components using the K-K relation, thus enabling minimally invasive tissue transparency in a tissue of live subjects. In a still further aspect, the disclosed method can be employed for achieving optical transparency and waveguiding in vivo, including tissues and organs that cannot be removed from living subjects. In another aspect, the disclosed method does not require performing a surgical procedure on a living subject to access tissue. In still another aspect, the disclosed method does not require use of toxic solvents, fixatives, or the like, in order to visualize tissues.

[0042] Previously, optical clearing has been demonstrated using intensely absorbing molecules. However, these molecules exhibit absorption in the blue region of the visible spectrum that blocks short wavelengths, thus achieving transparency only in the longer-wavelength red region. Furthermore, many of these molecules impart a color to specimens and organisms, thus potentially obscuring features of the underlying tissue. Thus, in one aspect, disclosed herein are methods of optical clearing using molecules that do not impart a color to specimens and organisms, and / or that achieve transparency in regions with different wavelengths such that the disclosed methods can be tuned based on the particular organs and / or tissues being observed. Optical Clearing in Tissues

[0043] In an aspect, light refraction and reflection occur at interfaces when refractive indices change. In another aspect, biological systems such as tissues are inhomogeneous media with different length scales and refractive indices. In still another aspect, reducing the refractive index mismatch between scatterers and background inside tissues can increase light transmission.

[0044] In another aspect, the pigments and non-pigment compounds useful in the disclosed methods are minimally toxic, have good water solubility, and are safe for topical administration. In another aspect, the pigments can diffuse or be injected into the biological tissues. In still another aspect, the disclosed method enables a significant increase in optical transmission in otherwise turbid biological tissue. In one aspect, the method can be conducted in live subjects, achieving transmission in various tissues without the need for invasive surgical procedures. Further in this aspect, complete recovery of the subjects after performing the method is observed. Methods for Tissue Clearing Using Clearing Compounds

[0045] In one aspect, disclosed herein is a method for imaging an organ or tissue in a subject, the method including:ATTORNEY DOCKET NO.221910-2720 (a) administering a composition that includes a clearing compound to the subject, wherein an interaction between the clearing compound and at least one overlying tissue in the subject creates a transparent spectral window in the at least one overlying tissue; and (b) visualizing the organ or tissue through the at least one overlying tissue; wherein the transparent spectral window comprises the entire visible spectrum.

[0046] In another aspect, the transparent spectral window can be from about 380 nm to about 780 nm, 400 nm to about 750 nm, about 400 nm to about 700 nm, about 500 nm to about 750 nm, or about 400 nm to about 600 nm.

[0047] In a further aspect, the organ can be a brain, a gastrointestinal tract, muscle, bones, blood vessels, a liver, a bladder, a spinal cord, a retina, a heart, a pancreas, a spleen, lungs, a trachea, kidneys, lymph nodes, a thymus, ovaries, testes, a uterus, a prostate or any combination thereof, or the tissue can be brain tissue, gastrointestinal tissue, muscle, connective tissue, bone tissue, blood vessels, liver tissue, bladder tissue, a tumor, spinal cord tissue, retinal tissue, heart tissue, pancreatic tissue, spleen tissue, lung tissue, tracheal tissue, kidney tissue, lymph node tissue, thymus tissue, ovary tissue, testes tissue, uterine tissue, prostate tissue or any combination thereof. In any of these aspects, the at least one overlying tissue can be skin, muscle, epithelial tissue, connective tissue, or any combination thereof.

[0048] In another aspect, individual neurons in a subject can be visualized without disruption of the scalp or skull of the subject. Further in this aspect, the disclosed method allows for noninvasive imaging of both YFP and the GFP-based calcium sensor GCaMP in the live mouse brain. In another aspect, the disclosed creation of a color-neutral transparency window in the mouse scalp allows repeated longitudinal imaging of the same brain regions in the same mouse throughout development.

[0049] In one aspect, the clearing compound can be selected from ampyrone, sunset yellow, Trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate; Sodium 1H-indene-1,3(2H)-dione- 2-(2- quinolinyl)sulfonate; 1,7-bis(4-hydroxy-3-methoxyphenyl)-(1E,6E)-1,6-Heptadiene-3,5-dione; Sodium 5-hydroxy-1-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazeny l]-1H-pyrazole-3- carboxylate ; Sodium 4-dimethylaminoazobenze ne-2′-carboxylate (methyl red); Disodium 6- hydroxy-5-[2-(4-sulfophenyl)diazenyl]-2-naphthalenesulfonate; Disodium spiro[isobenzofuran- 1(3H),9′-[9H]xanthen]-3-one- 2′,4′,5′,7′-tetrabromo-3′,6′-dioxide; Disodium spiro[isobenzofuran-ATTORNEY DOCKET NO.221910-2720 1(3H),9′-[9H]xanthen]-3-one- 2′,4′,5′,7′-tetraiodo-3′,6′-dioxide; Disodium 3,3′-([1,1′-biphenyl]-4,4′- diyl)bis(4-aminonaphthalene-1-sulfonate) (Congo red); Disodium 6-hydroxy-5-[(2-methoxy-5- methyl-4-sulfophenyl)azo]-2-naphthalenesulfonate; 9-(2-Carboxyphenyl)-6-(diethylamino)-N,N- diethyl-3H-xanthen-3-iminium chloride (Rhodamine B); 4-[2-[(2S)-2-carboxy-5-(β-D- glucopyranosyloxy)-2,3-dihydro-6-hydroxy-1H-indol-1-yl]ethenyl]-2,3-dihydro-(2S)-2,6- Pyridinedicarboxylic acid; 4,4′-(1,1-dioxido-3H-2,1-benzoxathiol-3-ylidene)bis[3-methyl-phenol; Sodium benzenemethanaminium N-[4-[[4-[(4-ethoxyphenyl)amino]ph enyl][4-[ethyl[(3- sulfophenyl)methyl]ami no]phenyl]methylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl-3-sulfonate; Tetrasodium 4-amino-5-hydroxy-3,6-bis[2-[4-[[2-(sulfooxy)ethyl]sulfonyl ]phenyl]diazenyl]-2,7- naphthalenedisulfonate; Disodium benzenemethanaminium N-ethyl-N-[4-[[4-[ethyl[(3- sulfophenyl)methyl]ami no]phenyl](2-sulfophenyl)methylene]-2,5-cyclohexadien-1-ylidene]-3- sulfonate; Sodium 4-[[4-(dimethylamino)phenyl] -(4-dimethyliminiocyclohexa-2,5-dien-1- ylidene)methyl]-3-hydroxy-7-sulfo-naphthalene-2-sulfonate; Sodium 2-[[4-(diethylamino)phenyl]- (4-diethyliminiocyclohexa-2,5-dien-1-ylidene)methyl]-4-hydroxy-5-sulfo-benzenesulfonate; 3,7- bis(diethylamino)-phenoxazin-5-ium chloride; 3,7-bis(dimethylamino)phen azathionium chloride (methylene blue); Sodium 4-[2-[2-[2-chloro-3-[2-[3,3-dimethyl-1-(4-sulfonatobutyl)indol-1-ium-2- yl]vinyl]cyclohex-2-en-1-ylidene]ethylidene]-3,3-dimethyl-indolin-1-yl]butane-1-sulfonate; or any combination thereof. In some aspects, the clearing compound is ampyrone or sunset yellow. In one aspect, the clearing compound is not tartrazine.

[0050] In an aspect, the subject is a mammal, a bird, a reptile, an amphibian, a fish, an arthropod, a mollusk, a cnidarian, an echinoderm, an annelid, a platyhelminthes, a nematode, or a plant. In one aspect, the mammal is a human, rat, mouse, rabbit, vole, tree shrew, guinea pig, hamster, cat, dog, pig, sheep, cow, or horse. In another aspect, the bird is a chicken, turkey, duck, parrot, or finch.

[0051] In any of these aspects, the organ or tissue is visualized in situ in the subject. In one aspect, in the disclosed method, performing step (a) reduces light scattering between two or more tissue components, the tissue components having different refractive indices. In another aspect, in the disclosed method, performing step (a) increases light transmittance through the at least one overlying tissue by at least 50-fold compared to light transmittance through the at least one overlying tissue before performing the method. In any of these aspects, performing the method allows visualization of at least one feature in the subject at least about 200 μm below a skin surface of the subject, or at least about 300, 400, 500, 600, or 700 μm below a skin surface of the subject.ATTORNEY DOCKET NO.221910-2720

[0052] In another aspect, an aqueous solution of the clearing compound having a concentration from about 30% (w / w) to about 45% (w / w), or about 35% (w / w) to about 40% (w / w), or about 38% (w / w) can be applied to an area of tissue to be cleared using a cotton-tipped applicator or by another means. In one aspect, the exact concentration will be determined by a number of factors including solubility of the compound in water, particular absorption properties of the compound, depth of visualization required, and the like. In an aspect, about 100 μL of solution are enough to treat about 1 cm2area of skin and the amount can be scaled accordingly to address larger or smaller areas for visualization as required. In some aspects, for longer visualizations, the composition including the clearing compound can be applied more than once such as, for example, every 10 to 15 minutes or as needed to maintain tissue transparency.

[0053] In one aspect, the composition is administered to the subject topically. In another aspect, the clearing compound is non-toxic. In a further aspect, following visualizing, the clearing compound is excreted by the subject in less than about 10 hours or in less than about 6 hours. In some aspects, a wipe or other means can be used to remove most or all of the clearing compound following visualizing. In another aspect, the clearing effect can last up to about 20 minutes before a gradual diminishment due to diffusion.

[0054] In any of these aspects, visualizing is accomplished using reflectance imaging, fluorescence imaging, laser speckle imaging, two-photon excitation spectroscopy, optical coherence tomography (OCT), light sheet microscopy, super-resolution microscopy, epifluorescence microscopy, fluorescence mediated tomography, photoacoustic tomography, three-photon microscopy, Brillouin microscopy, Raman microscopy, confocal microscopy, TIRF microscopy, brightfield / darkfield microscopy, DIC microscopy, structured illumination microscopy, or a combination thereof.

[0055] Also disclosed herein are pharmaceutical compositions including at least: (a) a clearing compound that interacts with at least one overlying tissue in a subject to create a transparent spectral window in the at least one overlying tissue; and (b) at least one carrier or excipient.

[0056] In a further aspect, in the pharmaceutical compositions the clearing compounds are as described above, while the at least one carrier or excipient can be selected from water, saline, sodium chloride, or other salts, a polysaccharide, an alcohol, a polymeric material, and other carriers and excipients known in the art for delivery of agents to tissues, for example, for theATTORNEY DOCKET NO.221910-2720 delivery of water soluble agents. In some aspects, the pharmaceutical compositions include at least one additional imaging agent.

[0057] Also disclosed herein are kits for achieving optical transparency in a living subject, the kit including at least a disclosed pharmaceutical composition and instructions for use thereof. In a further aspect, the living subject is a human or a laboratory animal. Mathematical Modeling of the Kramers-Kronig Relation

[0058] In one aspect, the real part (n) and imaginary part (k) of the refractive index of a material are related by the Kramers-Kronig (K-K) relation. In another aspect, in the frequency domain, the Kramers-Kronig relationship can be represented by the following equation:

[0059] In anotherin the wavelength domain as follows:

[0060] In still anotherindex (absorption of the material) the real part of the refractive index will have a nonlinear change in the neighboring wavelength. Further in this aspect, the refractive index in the longer wavelength will increase.

[0061] In one aspect, the real part and imaginary part of refractive indices of dye solutions and other clearing compound solutions can be measured by any technique known in the art, such as using an ellipsometer. In another aspect, the real part of the refractive index can be modulated by increasing the imaginary part accordingly.

[0062] In one aspect, disclosed herein is a method for imaging an organ or tissue in a subject, the method including at least the steps of (a) administering a composition comprising a compound to the subject, wherein an interaction between the compound and at least one overlying tissue in the subject creates a transparent spectral window in the at least one overlying tissue; and (b) visualizing the organ or tissue through the at least one overlying tissue. In another aspect, theATTORNEY DOCKET NO.221910-2720 transparent spectral window is in the visible region of the electromagnetic spectrum, such as, for example, from about 380 nm to about 780 nm.

[0063] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0064] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0065] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0066] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0067] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.ATTORNEY DOCKET NO.221910-2720

[0068] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0069] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0070] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions

[0071] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0072] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a dye,” “a salt solution,” or “an excipient,” include, but are not limited to, mixtures or combinations of two or more such dyes, salt solutions, or excipients, and the like.

[0073] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of theATTORNEY DOCKET NO.221910-2720 ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0074] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0075] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub- ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0076] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances,ATTORNEY DOCKET NO.221910-2720 conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0077] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a compound refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g., achieving the desired level of transparency of a given organ or tissue structure. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the condition being monitored, desired length of time of transparency, and any other concurrent or subsequent treatments to be performed.

[0078] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0079] As used herein, “overlying tissue” refers to a tissue in the body of a subject positioned over an organ or tissue that is desired to be visualized. The overlying tissue will typically have a different refractive index from the organ or tissue to be visualized and this can create scattering when standard visualization methods are attempted. In one aspect, application of a disclosed clearing compound or composition to the overlying tissue can create a transparent spectral window in the overlying tissue, allowing visualization of structures beneath. In one exemplary aspect, an overlying tissue could be skin, through which muscle is visualized, or an at least one overlying tissue could include both skin and muscle, through which bone, blood vessels, or neural tissue could be visualized. In any of these aspects, the overlying tissue can remain in place while visualization occurs, without the need for surgical intervention, laparoscopy, or the like.

[0080] A “clearing compound” as used herein refers to a compound that, when administered to a subject, renders at least one tissue transparent for a period of time.ATTORNEY DOCKET NO.221910-2720

[0081] As used herein, “administering” can refer to an administration that is topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intradermal, or any other useful means for clearing tissues and / or organs in an area of interest.

[0082] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form that may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0083] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form, which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

[0084] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g., human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0085] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed clearing compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0086] As used herein, “effective amount” can refer to the amount of a disclosed clearing compound or pharmaceutical composition provided herein that is sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term can also include within its scope amounts effective to enhance or restore toATTORNEY DOCKET NO.221910-2720 substantially normal physiological function.

[0087] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0088] The term “contacting” as used herein refers to bringing a disclosed clearing compound or pharmaceutical composition in proximity to a cell, a target protein, or other biological entity together in such a manner that the disclosed clearing compound or pharmaceutical composition can affect the activity of the a cell, target protein, or other biological entity, either directly; i.e., by interacting with the cell, target protein, or other biological entity itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the cell, target protein, or other biological entity itself is dependent.

[0089] It is understood, that unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere). Pharmaceutical Compositions

[0090] In various aspects, the present disclosure relates to pharmaceutical compositions comprising an effective amount of at least one disclosed clearing compound or a pharmaceutically acceptable salt thereof. As used herein, “pharmaceutically-acceptable carriers” means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, and adjuvants. The disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy and pharmaceutical sciences.

[0091] The compounds described herein are typically to be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The deliverable compound will be in a form suitable for topical administration, intravenous injection, or parenteral administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. The compounds may be administered as a dosage that has a known quantity of the compound.

[0092] Pharmaceutical compositions of the present disclosure can be in a form suitable for topicalATTORNEY DOCKET NO.221910-2720 administration. As used herein, the phrase “topical application” means administration onto a biological surface, whereby the biological surface includes, for example, a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus, and genital areas) or a mucosal membrane. By selecting the appropriate carrier and optionally other ingredients that can be included in the composition, as is detailed herein below, the compositions of the present invention may be formulated into any form typically employed for topical application. A topical pharmaceutical composition can be in a form of a cream, an ointment, a paste, a gel, a lotion, milk, a suspension, an aerosol, a spray, foam, a dusting powder, a pad, and a patch. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the present disclosure, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0093] The pharmaceutical composition (or formulation) may be packaged in a variety of ways. Generally, an article for distribution includes a container that contains the pharmaceutical composition in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, foil blister packs, and the like. The container may also include a tamper proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container typically has deposited thereon a label that describes the contents of the container and any appropriate warnings or instructions.

[0094] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere). Applications of the Disclosed Compositions and Methods

[0095] The scope of the invention encompasses various methods of use of the compositions disclosed herein. The scope of the invention encompasses a method of achieving optical transparency in a selected tissue of a subject by the use of or more optical clearing compositions disclosed herein. In various embodiments, the method of use may be performed in the context of research, performance of a diagnostic procedure, or to aid in the administration of a treatment or intervention, e.g. performance of a surgical treatment or the placement or guidance of an implant, endoscopic instrument, injection, or device.

[0096] The subject of the method may be a live animal, for example a human patient, or a non- human animal such as a research animal (e.g. mouse, rat, or non-human primate), a pet (e.g.ATTORNEY DOCKET NO.221910-2720 dog or housecat), and other animals including, but not limited to, livestock (e.g. cow, pig, chicken, or horse), exotic species, and wildlife. In other embodiments, the subject of the method may be a cadaver, an explanted body part, organ or tissue, or an organoid.

[0097] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. EXAMPLES

[0098] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. Example 1: Optical Clearing with Absorbing Molecules and Imaging in Live Mice Absorbing molecules make skin and muscle transparent in live mice

[0099] Tartrazine solutions and other dye solutions as described herein were applied topically to the scalp of a shaved, live mouse head and used laser speckle contrast imaging (LSCI) for visualizing cerebral blood vessels in the head. LSCI usually requires removal of the scalp due to its opacity, and imaging of the shaved mouse head did not reveal any interesting features of cerebral vessels. In contrast, after applying the dye solution, LSCI reveals structures representative of cerebral vessels. To confirm the location of these vessels in the brain, LSCI was performed after removing the scalp, identifying similar structures. Besides tartrazine, a few other absorbing molecules demonstrate a similar degree of scalp transparency (FIGs.3A-3F). Topically applied dye molecules can be removed from the scalp by rinsing with water, effectively reversing the transparency effect in a repeatable manner In addition to topical application, injecting dyeATTORNEY DOCKET NO.221910-2720 molecules into the scalp produces similar LSCI images, offering an alternative method for achieving tissue transparency in thicker skins.

[0100] It was next sought to demonstrate the ability of absorbing molecules to achieve tissue transparency in the mouse abdomen. Specifically, when a dye solution was topically applied to the abdominal skin of a live mouse under anesthesia and gently massaged on the skin, the abdominal skin not only darkened in color but also became more transparent in the spectral window associated with the dye. This transparency effect can be readily visualized with the naked eye and does not require any specialized imaging equipment. A few absorbing molecules have been proven to render the mouse abdomen transparent as described elsewhere herein. The transparent abdomen allowed direct observation of internal organs, including the liver, small intestine, cecum, and bladder. Moreover, their movements, such as peristalsis, as well as motions synchronized with the heartbeat and breathing could also be observed. The achieved abdominal transparency can be reversed by rinsing and massaging the skin with water. Physical mechanism of optical transparency in live tissues by absorbing molecules

[0101] Despite the strong absorption of tartrazine in the UV and blue region of the spectrum, in solution it remains minimally absorbing above 600 nm, even up to a concentration of 0.78 M. The high transmittance in the ≥600 nm spectrum agrees with the present observation that tissue transparency is primarily achieved in the red wavelengths. Therefore, it is speculated that the observed transparency stems from increased absorption at wavelengths shorter than those exhibiting transparency.

[0102] It is hypothesized that dye molecules with strong absorption and superior solubility can significantly increase the RI of the aqueous medium at wavelengths immediately longer than the absorption upon dissolution. This is confirmed with the Kramers-Kronig relations that connect the spectral dependences of the real (n') and imaginary (n'') components of the RI of a material through causality:value of the integral. This equation shows that the spectral location, spectral width, and absorption strength of a dye are all important parameters that control the achievable changes in n'(λ). As a numerical example, significant changes in the n' of the solution (on the order of 0.1) can be realized in the visibleATTORNEY DOCKET NO.221910-2720 spectrum by dissolving tartrazine molecules with a Gaussian absorption peak centered at 428 nm, a full-width at half-max (FWHM) of 94 nm, and a peak absorption coefficient of µa = 4πn''λ−1= 0.015 nm−1(corresponding to a value of n'' = 0.5). Consequently, the n' of the aqueous solution increases significantly at λ > 428 nm, reaching that of lipids and collagen fibers (n' = 1.43-1.53). Notably, n' stays significantly higher than that of pure water (n' = 1.33), even at wavelengths where n'' has essentially returned to zero and the absorption is negligible (e.g., >800 nm). The achievable changes in n' are proportional to the increase in n'', thus suggesting that water-soluble dye molecules with high n'' are effective agents to increase n' of the aqueous medium at wavelengths longer than the absorption upon dissolution.

[0103] These findings align with reported effects showing that absorbing molecules can increase the RI of a medium when dissolved or dispersed within it. For instance, water-soluble food dyes (e.g., Brilliant Blue, Allura Red, Pyranine, and Quinoline Yellow) have been shown to significantly alter the RI of aqueous solutions upon dissolution. Similarly, plant pigments (e.g., chlorophylls, carotenoids, and anthocyanins) can enhance the RI of commercially available polymers when dispersed within their matrices. Additionally, hematoxylin and eosin stains are noted for changing the RI of intracellular components. However, none of these studies have explored the use of absorbing molecules to reduce scattering in their systems, let alone achieve tissue transparency in vivo. Apart from these “RI engineering” studies, observations have shown that naturally occurring, strongly absorbing pigments in apple skin can influence light scattering by altering the RI. It is noteworthy that this study does not actively utilize apple skin pigments to alter optical properties but instead highlights how nature utilizes the Kramers-Kronig relations.

[0104] Optimizing the spectral properties of a dye molecule would enable optical transparency at a concentration that is both low and physiologically tolerable. To this end, optical behavior of the dye molecule was simulated using the Lorentz model with a single optical resonance. Herein it is shown that the multi-oscillator Lorentz model can be reduced to a single-oscillator model for molecules with multiple absorption peaks. With this model, the relative permittivity εr(and complex refractive index n = √^^r) of the OCA can be directly linked to its plasma frequency ωpand damping constant γ:ATTORNEY DOCKET NO.221910-2720

[0105] Here, ε∞ is the high-frequency dielectric constant, γ is approximately equal to the full-width at half-maximum (FWHM) of the resonance peak. Choosing an operating frequency ω in the vicinity of a resonance at ω = ω0− Δω simplifies the expression to:of an absorbing molecule in raising n' of the medium it is dissolved in. First, given the ω0−1scaling, absorbing molecules with low resonance frequencies (i.e., small ω0) are more effective in raising n' of the medium. It is also strategic to choose a resonance frequency that lies just above the frequency used for imaging (i.e., a small positive-valued Δω). This ensures that the maximum increase in the permittivity is obtained due to the resonance without a detrimental amount of absorption. Since optical imaging is usually performed in the visible spectrum, these requirements suggest that dyes with absorption peaks in the blue region of the spectrum make more effective OCAs than those with absorption peaks in the near-UV (NUV, 300-400 nm) and shorter wavelengths. Second, the absorbing molecule should have a low damping constant γ, which is reflected in its absorption spectrum as a narrow peak with a small FWHM. Eq. (3) predicts that a dye molecule with sharp absorption peaks act as a more intense Lorentz oscillator with a larger εrvalue near its resonance. It is also important to avoid significant attenuation due to absorption at the imaging wavelength. For the strongly absorbing dye considered here, it is thus important to stay a bit away from the resonance. Empirically, it has been found that the dye molecules used herein are operated at ω = ω0− γ to achieve sufficient transparency.

[0107] The Kramers-Kronig relations (Eq. (1)) and the Lorentz model (Eqs. (2) and (3)) predict that as the resonance absorption wavelength of absorbing molecules increases, they become more effective in raising n' of the medium. To illustrate this point, three Lorentz oscillators were modeled with their resonances at 100 nm, 250 nm, and 400 nm (FIGs. 1B-1C). The Lorentz resonator at 100 nm exhibits weak absorption in n'', leading to a small increase in n' in the visible spectrum. In contrast, the Lorentz resonator at 400 nm is predicted to be a stronger absorber with a higher peak n'', thus resulting in a more efficient increase in n' at longer wavelengths. This theoretical prediction is validated experimentally, as evidenced by the small molar absorption coefficient α of 0.012 M−1at 84 nm, of the common OCA, glycerol, corresponding to a small molar ∆n' coefficient, β (defined as the change in n' of the medium induced per molar concentration of dissolved OCA), of 0.0129 M−1in the visible spectrum upon dissolution in water (FIGs.1D-1E andATTORNEY DOCKET NO.221910-2720 Table 1). This finding reveals that conventional OCAs, such as glycerol, raise the RI of tissue because they are themselves absorbing molecules in the short-wavelength, extreme UV (EUV) spectrum (usually < 150 nm). However, because they absorb at extremely short wavelengths (likely intended to be completely transparent in the visible spectrum), conventional OCAs are not strong absorbers and thus inefficient at raising n' of the medium. For example, with a small β of 0.0129 M-1for glycerol, one must use glycerol at a concentration of 11.6 M (almost pure glycerol), to raise the baseline n' of water to match that of lipids. This requirement, which is deeply rooted in physics, explains why glycerol necessarily results in dehydration and shrinking of tissue when applied as an OCA. Table 1: Efficiency of Absorbing Dye Molecules in Achieving Optical Transparency Compared to Conventional Optical Clearing Agents

[0108] This theoretical prediction also agrees with the α and β spectra of tartrazine (FIGs.1F-1I and Table 2), with a high absorption coefficient of 2.04 ± 0.02 × 104M−1cm−1at its peak absorption of 428 nm, while becoming completely transmissive beyond 600 nm (FIG.1J). This dependence of the RI increase on the resonance frequency is further confirmed by plotting α and β of 23 absorbing molecules vs. their peak absorption from 84 nm to 800 nm, with stronger absorption and a more efficient n' increase at longer wavelengths (FIGs. 1K-1L and Table 2). These 23 molecules include 21 absorbing molecules in the visible spectrum, along with the common OCAs glycerol and antipyrine, the latter of which absorbs in the short-wavelength UV spectrum. Antipyrine represents one of the best performing n'-matching agents after screening >1,600 chemicals; however, it has a much lower β than that of the visible-absorbing molecules, confirming the wavelength dependence of the achievable n' increase imposed by the Lorentz oscillator model.ATTORNEY DOCKET NO.221910-2720 Table 2: Exemplary Absorbing Dye Molecules Dye IUPAC Name Chemical Structure <α> <β> Index (M-1) (M-1)ATTORNEY DOCKET NO.221910-2720 Disodium 6- 0.028 0.10 hydroxy-5-[2-(4- sulfophenyl)diazenATTORNEY DOCKET NO.221910-2720 9-(2- 0.20 0.16 Carboxyphenyl)-6- (diethylamino)-N,N- ×ATTORNEY DOCKET NO.221910-2720 Tetrasodium 4- 0.19 0.12 amino-5-hydroxy- 3,6-bis[2-[4-[[2-ATTORNEY DOCKET NO.221910-2720 18 Sodium 2-[[4- 0.30 0.020 (diethylamino)phen yl]-(4- m

[0109] The Lorentz oscillator model, along with the Kramers-Kronig relations, predicts that optically absorbing molecules can bring about more substantial and desired changes in the optical properties than conventional RI-matching agents. According to the dependence revealed in Eq. (3) and FIGs. 1K-1L, the most efficient agent should have its peak absorption at the longest possible wavelength, which is upper-bounded by the imaging wavelength, to maximize n'' and ∆n'. At the same time, the agent should have a single narrow absorption peak without any additional absorption at the imaging wavelength. In addition to these purely optical requirements, the agents also need to display a high solubility and diffusivity in water as well as excellentATTORNEY DOCKET NO.221910-2720 biocompatibility to facilitate in vivo application. After sampling 21 candidates (FIGs.1M-1N and 4A-4E and Table 2), tartrazine emerges as the standout candidate that satisfies all requirements for optical transparency in the visible spectrum (FIG.1N). Specifically, tartrazine has a narrow absorption peak near the short extreme of the visible spectrum, and its solution is minimally absorbing above 600 nm (FIG.1J). In addition, it exhibits the highest β while maintaining a low α in the visible spectrum (FIG.1M), as evidenced by a “clear window” that appears at the shortest wavelength in the β / α spectrum for all molecules (FIG.1N).

[0110] The dye screening also reveals several other important findings. First, 14 dyes other than tartrazine offer similar or even greater potential for achieving tissue transparency at longer wavelengths, with Dye-21 showing the greatest potential in the NIR spectrum (FIGs.1L and 1N). Second, in vivo optical transparency is predicted to be more readily achieved in the NIR spectrum, owing to the exceptionally high values of β attained by resonant absorbers at longer wavelengths. Specifically, Dye-21 in FIG.1L exhibited a high β of >1.2 M−1at 800 nm, which is ~100x more efficient than glycerol and similar OCAs (Table 1), as predicted by the Lorentz oscillator model in Eq. (3).

[0111] Finally, the Lorentz oscillator model also provides theoretical foundation for the long- standing challenge associated with achieving in vivo tissue transparency. Despite recent advances in tissue clearing techniques, most are effective in ex vivo tissues, with very few applicable in vivo (Table 3). The present theoretical framework, grounded in the Lorentz oscillator model and Kramers-Kronig relations, offers insights into this challenge. Conventional OCAs such as glycerol, sucrose, and benzyl alcohol / benzyl benzoate (BABB) function as high-RI agents, bridging the RI disparity between water and other high-RI components (e.g., lipids and proteins) in endogenous tissues. However, the theoretical analysis shows that these molecules derive their high RI from absorption in the short-wavelength, EUV spectrum (FIGs.1A-1N). Eq. (3) predicts that these molecules are weak Lorentz oscillators, characterized by an extremely small β (Table 1). This means they inefficiently raise the RI of water per unit concentration when dissolved. This insight elucidates why these OCAs are typically used at very high concentrations (e.g., 80% glycerol or 90% sucrose) or in their pure forms (e.g., pure glycerol). Consequently, conventional tissue clearing necessitates almost complete substitution of the original water content with the OCA, leading to significant dehydration and shrinkage of the biological tissue. Such requirements make these traditional ex vivo tissue clearing methods unsuitable for use in live biological tissues. Table 3: Comparison among Absorbing-Molecule-Enabled Optical Transparency and Other Existing Tissue Clearin and In Vivo O tical Ima in MethodsATTORNEY DOCKET NO.221910-2720 Application Preservation Representative Time to Penetration Resolution of clearing optical depth ≥ 1 down to endogenous agents transparency mm in μm? %Validation of the mechanism enabling optical transparency via dye molecules

[0112] Increasing the absorption of a scattering medium has been shown to improve imaging resolution by suppressing scattered photons that contribute to the background noise. These results, on the contrary, are not due to the absorption of scattered photons by dye molecules, but originate from reduced scattering events in the tissue. To this end, conducted experiments were conducted using tissue-mimicking scattering phantoms. These scattering phantoms were produced by uniformly mixing silica spheres with an n' of 1.43 (close to that of lipids and collagen) in an optically transparent hydrogel with a background n' of 1.33 (the same as water). Agarose, used at a concentration of 6 mg / mL to solidify water into a hydrogel, has a minimal effect on the refractive index of the medium. The density of spheres was optimized to reach the same magnitude of the scattering coefficient of muscle tissue of ~10 cm−1. Wave optics simulations based on the finite-difference time-domain (FDTD) technique show how the particles scatter light and disturb the propagation of the incident planar light waves. However, when the n' of theATTORNEY DOCKET NO.221910-2720 hydrogel background was increased to match that of the scatterers, the incident wave travels without a noticeable distortion despite the presence of silica particles.

[0113] The scattering phantom is placed on top of a graphing paper showing a regular grid of millimeter-sized squares. Upon increasing the absorbance of this phantom by dissolving tartrazine into the hydrogel, the opaque phantom gradually turns transparent in the red part of the spectrum (λ ≥ 600 nm) and appears visually similar to the hydrogel without any scatterers. The effect of optical transparency was quantified by measuring the normal-incidence light transmittance (T) through the phantom as a function of dye concentration. In the red transparency window (λ ≥ 600 nm), an over-60-fold gradual increase of T was achieved with an increasing tartrazine concentration. At this concentration, the n' of the dye-doped hydrogel matches that of the silica spheres while producing a negligible n'' in the ≥600 nm range. Further increases in the dye concentration lead to a decrease in maximum transmittance, which is attributed to an overshoot in n' that causes an increase in scattering. These observations differ from the enhancement in image resolution induced by absorption; the latter displays a decrease in overall transmittance without exhibiting the overshoot behavior. The T measurements enable extraction of the scattering coefficient µs using the Bouguer-Beer-Lambert law:µt(µt= µa+ µs≈ µssince µs>> µa) for wavelengths beyond 600 nm. Normalized µtof the scattering phantom composed of increasing concentrations of tartrazine was plotted. A significant reduction in µt / µt,0Mis found for scattering phantoms with a high concentration of tartrazine beyond 600 nm, indicating a reduction in light scattering as a result of n' matching. Using dark-field microscopy, the observed reduction in scattering was verified not to be due to the aggregation of silica scatterers. Moreover, µt / µt,0Mwas plotted at varying wavelengths as a function of the n' of the aqueous background of the scattering phantom, which was doped with different dye concentrations. Importantly, these plots exhibit good agreement with a reported monodisperse scattering model, thus confirming the RI modification by water-soluble dye molecules via the Kramers-Kronig relations as the origin of improved transmittance of the phantoms beyond 600 nm.

[0114] The mechanism of optical transparency achieved by absorbing dyes was validated using real biological tissue, such as chicken breast, as the scattering medium. The chicken breast has a µtof 32.5 ± 3.5 cm−1at 600 nm, similar to the reported µs of ca.30 cm-1at 600-700 nm. In theATTORNEY DOCKET NO.221910-2720 transparency window in the red wavelengths (i.e., λ ≥ 600 nm), a gradual increase in T was achieved with an increasing concentration of tartrazine. Moreover, a significant reduction in µt / µt,originalis found for the chicken breast with an increasing concentration of tartrazine beyond 600 nm, indicating a reduction in light scattering as a result of n' matching rather than the absorption of scattered photons. A representative micrograph of the muscle section shows that tartrazine molecules penetrate myofibers and fill the aqueous sarcoplasmic space between the myofibrils. Consequently, tartrazine increases the refractive index of the sarcoplasm (1.35-1.37) to match that of the myofibrils (1.39~1.47), resulting in the optical transparency of the muscle tissue.

[0115] Lastly, the diffusion rates of tartrazine through common tissue types were measured to evaluate its potential as a generic OCA. It has been reported that the dynamics of fluid diffusion within biological tissue can be approximated by free diffusion. By tracking the temporal evolution of optical transmission during dye immersion, the diffusion coefficients of tartrazine molecules were extracted in the range of 0.067 ~ 0.66 × 10-6cm2s-1in various tissues including the mouse skin, mouse muscle, rat skin, and chicken breast, using a reported method. This range of diffusion coefficient is significantly higher than other commonly used OCAs (e.g., propylene glycol has been reported to have a small diffusion coefficient of 0.021 × 10-6cm2s-1). Consequently, tartrazine takes much shorter time to achieve optical transparency (e.g., 2 h for the 2 mm chicken breast tissue) than the required immersion time for other OCAs (e.g., 24 h for 60% sucrose) to achieve the same effect. This difference in diffusion rate is attributed to the much higher molar Δn' coefficient, β, of tartrazine than that of conventional OCAs (Table 1). As a result, tartrazine can achieve the desired Δ^^′ at a much lower concentration than conventional OCAs, leading to a more dilute solution with lower viscosity, which enhances its diffusion kinetics. Dynamic optical transmission measurements reveal that these tissues regain their original opacity as tartrazine molecules diffuse out, demonstrating the reversibility of the induced optical transparency. Dye molecules can improve the resolution of microscopic imaging within a scattering medium

[0116] Dye molecules can improve the spatial resolution in imaging at substantial depths in tissue-mimicking scattering media. The resolvable spatial frequencies of a 1951 US Air Force (USAF) resolution target were quantified when imaging it through 1 mm scattering phantoms with different tartrazine concentrations. The scattering phantom is placed between the objective lens and the USAF target, which is back-illuminated with a white lamp. Micrographs through the clear phantom without any silica particles reveal the smallest features with a line width of 2.19 μm.ATTORNEY DOCKET NO.221910-2720 However, the imaging resolution is greatly impacted in the presence of the scattering phantom. The spatial resolution progressively increases as the dye concentration in the phantom is increased, until clear resolution of the 2.19 μm line width. This gradual increase in spatial resolution is quantified through a measured increase in the modulation transfer function (MTF) across all considered spatial frequencies and wavelengths. The MTF displays a wavelength- dependent behavior. At shorter wavelengths, images become blurry once surpassing an optimum concentration, whereas at longer wavelengths, the impact on both MTF and spatial resolution reaches a plateau.

[0117] This wavelength-dependent behavior is consistent with the spectrally-dependent changes in the RI as a result of dye-doping. At shorter wavelengths, the real index of the aqueous (background) component n'backgroundcan overshoot the index of the silica particles. At longer wavelengths, the weaker dependence of the index enables accurately dialing in of n'backgroundto achieve transparency across a broad wavelength range. When the MTF at 101.6 line-pair per millimeter (lp mm-1), which is relevant for imaging micron-sized structures, is plotted against the difference between the modulated background real index (n'background) and the particle real index (n'particle), the data at all wavelengths collapse to the same master curve. This master curve, along with the transparency effect primarily in the red part of the spectrum (λ ≥ 600 nm), validates RI modification by water-soluble dye molecules via the Kramers-Kronig relations. The MTF was analyzed at various depths in the scattering phantom and found that 0.6 M tartrazine can resolve micron-sized features even at a depth of 3 mm. In contrast, scattering phantoms with pure water and 0.6 M glycerol could not resolve these features beyond 1 mm. MTF analysis for chicken breast tissue of different thicknesses revealed comparable performance by 0.6 M tartrazine, resolving features as small as 5 µm at depths beyond 1 mm. Similarly, the USAF resolution target became resolvable underneath various mouse tissues in a tartrazine solution, thereby consolidating the observed transparency effects in live mice.

[0118] Lastly, numerical, wave-optics simulations were carried out to model the interaction of an incident beam with scattering phantoms doped with and without tartrazine. After the 600 nm light beam propagates through a 1-mm-thick scattering phantom, the beam is refocused by an ideal lens; comparison of the point spread function highlights that a sharp focus can be achieved in the presence of tartrazine, in contrast to the original blurred focus in water. The simulations reveal the reduced scattering of 600 nm light waves by tartrazine due to the RI modification following the Kramers-Kronig relations, rather than the removal of scattered photons by absorption, as the mechanism of the observed optical transparency. Taken together, these data demonstrate theATTORNEY DOCKET NO.221910-2720 ability to resolve spatial frequencies up to 228.1 lp mm-1(i.e., line widths down to 2.19 μm) through millimeters-thick scattering phantoms that optically mimics biological tissue. Monitoring gut motility through a transparent abdomen using dye molecules

[0119] This investigation assessed whether the optical transparency would enable obtaining high-resolution structural and functional information from deep-seated tissues that typically resist noninvasive microscopic imaging in live animals. The enteric nervous system (ENS) was a focus, populating the gastrointestinal (GI) tract situated deep within the abdominal cavity. The ENS controls GI functions such as motility, luminal secretion, and absorption. Dysfunctions in the ENS are implicated in GI disorders such as irritable bowel syndrome and constipation, as well as the GI manifestations of neuropsychiatric illnesses such as autism and Parkinson’s disease. Neuronal cell bodies of the ENS are grouped in clusters (ganglia) and are found within two mesh-like layers in the GI tract wall: the submucosal plexus and the myenteric plexus. The myenteric plexus, positioned between the circular and longitudinal muscle layers, contains local reflex microcircuits that sense intestinal distension and initiate peristalsis, the rostro-caudal propulsion of luminal contents. Despite the ENS’s significance, the understanding of how myenteric neurons orchestrate gut motility is limited. Direct imaging of myenteric neurons during gut motility in live animals is hindered by the opaque nature of the abdominal wall. Existing techniques to monitor gut motility often involve placing a dissected tissue in an ex vivo organ bath or require the invasive surgical implantation of an abdominal window. The latter approach not only restricts normal gut movement but can also lead to colonic obstruction. These limitations underscore the need for non-invasive methods to image the movement of the GI tract and the ENS in their most native state in live animals.

[0120] Optical transparency was achieved in the abdomen of live choline acetyltransferase (ChAT)-Cre-tdTomato mice. ChAT-Cre-tdTomato mice express tdTomato specifically in cholinergic neurons, which are excitatory within the ENS. Fluorescence microscopic imaging of the myenteric plexus in the jejunum was then performed (FIG.2A), which is situated 500~800 μm beneath the skin’s surface. In untreated animals, no discernible images could be obtained at this depth range due to the scattering of overlying tissues (FIG.2B). However, the dye molecules effectively rendered the skin and muscle above the jejunum transparent, allowing direct visualization of the cholinergic neurons, along with their nerve fibers with <10 µm diameters, labeled by the red fluorescent protein, tdTomato, in the myenteric plexus (FIG. 2C). The fluorescence origin from jejunum neurons was confirmed by dissecting the overlying skin, muscle,ATTORNEY DOCKET NO.221910-2720 and jejunum for ex vivo imaging. Unlike the abdominal window, the “transparent abdomen” achieved using dye molecules does not involve any exogenous rigid materials that restrict natural gut movement. Consequently, the rapid dynamic movement of the myenteric plexus could be monitored—reflecting the underlying motility of the jejunum—at a high frame rate of 28 frames per second (fps; FIG.2D).

[0121] Particle image velocimetry (PIV), a technique commonly used to visualize the flow of fluids seeded with tracer particles, was employed to analyze the dynamic movement of the myenteric plexus, using tdTomato-labeled myenteric neurons as “landmarks” to assist in the PIV analysis. Specifically, by conducting a PIV analysis on the video and excluding frames associated with global movements due to heartbeat and breathing, a time-evolving vector map was generated. In this map, each subregion is assigned a vector that signifies the orientation and magnitude of tissue movement (FIG. 2D). The temporal evolution of the global movement pattern of the myenteric plexus was first investigated by calculating the average moving direction and magnitude (FIG. 2E). This global movement pattern reveals a periodic change in the moving directions coupled with occasional bursts of large movement magnitude. To visualize such periodic movement, a representative time series highlighting different moving directions and magnitudes was selected (FIG.2F). These time-evolving vector maps exhibit several informative findings. First, there exist periodic oscillations of global translation directions and switching between relatively featureless low-displacement maps and those with obvious movements (FIG. 2F). Second, various subregions within the field of view display patterns of tissue movement that deviates from unidirectional translation (FIG.2G). For instance, contraction (FIG.2H), expansion (FIG.2I), and rotation (FIG.2J) were observed in representative examples. To highlight these local movement patterns, the divergence of the vector map was computed to pinpoint regions that act as “drains” and “sources” for tissue movement in contraction (FIG.2H) and expansion (FIG. 2I). Similarly, the curl of the vector map was also computed for visualizing the rotation center (FIG. 2J). Third, the diversity of these movement patterns, all co-existing in a larger field of view at the same time, effectively rules out the possibility that they are solely due to the mouse’s breathing and heartbeat. These physiological processes would typically induce a global, vertical translational motion across the entire field of view. This approach offers the capability to visualize the activity of deep-seated tissues in live animals in their most native state. DiscussionATTORNEY DOCKET NO.221910-2720

[0122] Herein is reported a counterintuitive finding: strongly absorbing dye molecules, when applied topically to biological tissues, can reduce the intrinsic light scattering within these tissues. This effect renders various biological tissues—including the scalp, muscle, and abdomen— transparent. A theoretical framework was constructed based on the Lorentz oscillator model to explain this phenomenon. This model predicts that molecules with absorption at low resonance frequencies (i.e., long wavelengths) are more effective at raising the refractive index of water when dispersed than those with resonances at higher frequencies. To corroborate this hypothesis, extensive experiments and analyses were conducted using both tissue-mimicking scattering hydrogels and ex vivo biological tissues. These tests confirmed the mechanism behind these observations, and showcased the achievable spatial resolution through millimeters of scattering medium once transparency is attained.

[0123] Several limitations remain for this approach at its current stage. Specifically, this approach, like other similar in vivo tissue clearing methods, is based on matching the RI of the aqueous media to that of the lipids and proteins. However, since the RI of these lipids and proteins is heterogeneous (ranging from 1.4 to 1.5), it remains challenging to find a single, optimal concentration of the dye solution for perfect RI matching in a given tissue. Therefore, the observed optical transparency in ex vivo and in vivo tissues results from reduced, but not completely eliminated, scattering through them. In addition, since RI matching results from absorbing molecules dissolved in the aqueous medium, the achievable penetration depth depends on the diffusion of these molecules within a reasonable time frame. For thicker tissues, the injection of absorbing molecules offers an alternative method for achieving tissue transparency at greater depths.

[0124] Our theoretical framework predicts the possibility of identifying even much more efficient OCAs by searching for or synthesizing dye molecules that absorb in the NUV and blue regions of the spectrum. While this may seem counterintuitive, such molecules function as Lorentz oscillators with more intense resonance strength, thus yielding a higher efficiency in matching the RI in biological tissue on a per molar basis. Contrasting this with conventional clearing agents, tartrazine is >15x more efficient at RI matching in biological tissues, while Dye-21 is >100x more efficient at achieving the RI matching in the NIR spectrum (Table 1). Consequently, tartrazine can achieve in vivo optical transparency at safe concentrations with acceptable osmolarity when applied topically. Molecules with lower resonance frequencies, sharper absorption peaks, and richer delocalized electrons are expected to be more efficient than tartrazine in achieving optical transparency in living tissues.ATTORNEY DOCKET NO.221910-2720 Example 2: Materials and Methods for Additional Dye Molecules Chemicals and materials

[0125] Dye molecules used in this study are listed with their full IUPAC names, chemical structures, and sources in Table 2. Hydrochloric acid (HCl, 36.5-38.0 w / w%) and nitric acid (HNO3, 68.0-70.0 w / w%) were purchased from Fisher Scientific Company L.L.C. (Pittsburgh, Pennsylvania, USA) and used without further purification. The low melting temperature SeaPlaqueTM agarose was purchased from Lonza (Basel, Switzerland). Silicon dioxide spheres (powder; 637238) were purchased from Sigma-Aldrich (Burlington, Massachusetts, USA). Silica sphere suspension (SISN1000) was purchased from nanoComposix (San Diego, California, USA). The 1X phosphate-buffered saline (PBS) was purchased from Gibco (Waltham, Massachusetts, USA). Water used in this work was purified by a Millipore Milli-Q Integral 10 water purification system.

[0126] For transmission measurements of scattering phantoms, the phantom with the mold was placed in the light path of the UV-Vis spectrometer. A clear hydrogel without silica scatterers in the mold was used as the reference to correct for any attenuation due to baseline absorption and reflection at the air-hydrogel interfaces.

[0127] For transmission measurements of ex vivo tissues, the tissue sample was first mounted on a microscope slide (precleaned, 12-550-15, Fisher Scientific), which was placed in the light path of the UV-Vis spectrometer to ensure that the entire incident beam was blocked by the tissue. For dynamic UV-Vis experiments, the samples were attached to one side of a 1 cm path cuvette holding 3 mL of the dye solution and time-elapsed measurements were carried out with a time interval of 5 minutes until the transmission reached a plateau. Mass spectrometry

[0128] Dyes were dissolved with water to a concentration of 0.1 mg / mL and this solution was analyzed by LC-ESI / MS on the Waters Acquity UPLC and Thermo Exploris 240 BioPharma orbitrap mass spectrometer. Spectra were collected in positive full scan mode, mass range of 150-2000 Da, Orbitrap resolution 120000. The column was a Phenomenex Kinetex F52.6u 100A 150x2.1 mm with mobile phases 0.1% formic acid in water and 0.1% formic acid in acetonitrile in a gradient mode at a flow rate of 0.25 mL / min. The injection volume was 3 µL. Preparation and imaging of hydrogel−silica scattering phantomsATTORNEY DOCKET NO.221910-2720

[0129] The as-received silica particles were characterized by an Apreo S LoVac scanning electron microscope (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and NanoBrook Omni particle size and zeta potential analyzer (Brookhaven Instrument, Holtsville, New York, USA) before use. The hydrogel−silica scatter phantoms were prepared by first mixing the low melting temperature agarose and silica particles with water or tartrazine solutions of different concentrations. The final concentration of agarose and silica particles in the mixture was 6 mg mL−1and 10 mg mL−1, respectively. The mixture was then heated in an oven at 70 °C for at least 10 min to facilitate the dissolution of agarose. A 1-mL suspension was pipetted into a plastic base mold (15 × 15 × 5 mm, 22-363-553, Fisher Scientific). The solution was refrigerated at 4 °C for 10 min for solidification. The scattering phantom was then placed on a home-printed transparent pattern illuminated by a white LED panel for imaging.

[0130] Other hydrogel−silica scattering phantoms were prepared by mixing the silica particle suspension (1 μm, 10 mg mL−1) with low melting temperature agarose and the optical clearing agents at specific concentrations. The final concentration of agarose was 6 mg mL−1for all phantoms. The mixture was then heated in an oven at 70 °C for at least 10 min to facilitate the dissolution of agarose. A suspension of the mixture was pipetted and injected to fill the well of a silicone isolator with varying depths (JTR20R-A2 series, Grace bio-labs, Bend, Oregon, USA) mounted on a coverslip (24 × 40 × 0.15 mm, 12-544-C, Fisher Scientific). The top of the solution was sealed by another coverslip (22 × 22 × 0.15 mm, 3306, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and the phantom was left at room temperature for slow solidification. The phantoms were then placed on top of the negative 1951 USAF resolution test target (R3L3S1N, Thorlabs, Newton, New Jersey, USA) and imaged using an optical microscope (DM2700M, Leica Microsystems, Wetzlar, Germany) with a 10X objective (numerical aperture: 0.25, N Plan Epi BD, Leica Microsystems). White-light illumination was generated by an arc lamp (66921, Newport, Irvine, California, USA). Filters selecting different wavelength ranges were inserted in the front of a scientific CMOS camera (C11440, ORCA-Flash4.0, Hamamatsu Photonics, Japan). Bandpass filters centered at 525 nm (FF01-525 / 45-25), and 785 nm (LD01-785 / 10-25) were purchased from Semrock (Rochester, New York, USA). Bandpass filters centered at 600 nm (FB600-10) and 680 nm (FB680-10) were purchased from Thorlabs. Dark-field microscopy of silica particles

[0131] To visualize the spatial distribution of silica particles in the agarose gel, dark field images were captured using a Leica DM2700M microscope equipped with a dark field objective (N PLANATTORNEY DOCKET NO.221910-2720 EPI 10x / 0.25 BD, Leica) and CMOS camera (ORCA-Flash4.0 LT3 Digital CMOS camera, Hamamatsu). Centroids of individual particles were tracked using algorithms previously developed for analyzing digital micrographs of colloidal particles. Calculation of the modulation transfer function (MTF)

[0132] The MTF measures the ability of an imaging system to reproduce contrast for various spatial frequencies. It represents how the system captures the detailed structure of an image based on the spatial frequency of line or sine wave patterns, which are measured in line pairs per millimeter (lp mm-1), as demonstrated by the 1951 USAF target. The MTF was determined by calculating the normalized difference between the maximum and minimum transmitted light intensities to assess contrast modulation:of the bars in the USAF target. The MTF calculation in this work is specifically tailored for a set of spatial frequencies supported by the USAF target. Preparation of ex vivo chicken breast tissue samples

[0133] Raw boneless and skinless chicken breast was bought from local sources and kept frozen at 20 °C. Before experiments, the frozen, raw chicken breast was allowed to thaw at room temperature for 10 min. The chicken breast was then sliced to the desired thickness (0.5 mm to 5 mm) parallel to collagen fibers to ensure that each piece has fixed muscle fiber orientation. The thickness of the tissue was measured via a digital caliper (500-196-30, Mitutoyo) by sandwiching the tissue between two coverslips (22 × 22 × 0.15 mm, 3306, Thermo Fisher Scientific). The ex vivo tissue sample was sequentially soaked in a 20 mM tartrazine solution for 20 min, a 163 mM tartrazine solution for 20 min, and a 0.62 M tartrazine solution for 2 h to yield tissue samples of different tartrazine concentrations. To facilitate the soaking of the sample in the tartrazine solution, the sample was placed on an orbital shaker (SHKE4000-7, Thermo Scientific) at 100 rpm and 40 °C. Ellipsometry measurements of the RI of dye solutions

[0134] A Horiba UVISEL ellipsometer was used to measure the real and imaginary parts of the RI of solutions containing different concentrations of a specific dye molecule. Specifically, 5 mL of the solution was added to a shallow petri-dish (35 mm diameter × 10 mm height, CorningATTORNEY DOCKET NO.221910-2720 Falcon, New York, USA). The solution covered the entire bottom of the petri-dish, creating a flat, reflective air−liquid interface. Reflected light was measured at an angle of 69.85° and the RI of the solution was calculated with a single interface semi-infinite reflection model. For all measurements, a 0.2 s integration time was applied at each wavelength. Measurements of the molar n' change, β

[0135] The molar n' change, β, is defined and measured according to the following quantitative relationship between the n' and c (i.e., the concentration) of a certain molecule dissolved in an aqueous solution: where n'wis the real RI of pure water, c is the molar concentration (in mol L−1, or M) of the dissolved molecule, and β is its molar n' change (in M−1). Note that n', n'w, and β all have wavelength dependence. According to Eq. (6), by measuring the n'(λ) of the solutions with ellipsometry, one can obtain β(λ) by fitting n'(λ) with c to a linear relationship.

[0136] The importance of β lies in its ability to guide the selection of appropriate dye molecules as OCAs and the optimization of their concentrations to reduce scattering in biological tissues. For imaging at a specific wavelength of interest, λ0, one should select dye molecules with the highest β(λ0) and lowest α(λ0). Since light scattering in most tissues (skin, muscle, brain, etc.) is primarily due to a difference in the real RI between water (1.33) and lipids / proteins (1.4~1.5), the required concentration of the chosen dye molecules can be approximately determined by calculating (1.45-1.33) / β(λ0). This formula, however, merely provides a preliminary estimate, since the RI of lipids and proteins varies; thus, a singular dye concentration that perfectly matches the RI for any tissue does not exist. Consequently, precise concentration determination requires experimental optimization. UV-Vis transmission spectroscopy

[0137] A Thermo Fisher Scientific Evolution 350 UV-Vis spectrophotometer was used to measure the transmission and absorption spectra of dye solutions, silica scattering phantoms, and ex vivo tissues. For all measurements, a 0.2 s acquisition time was applied at each wavelength. The wavelength range was 190-1100 nm during all measurements.

[0138] For transmission measurements of dye solutions, a 1 mm optical path quartz cuvette (high precision, two polished sides, Orient Analytics, Yixing, China), a 0.1 mm optical path quartzATTORNEY DOCKET NO.221910-2720 cuvette, and a 0.01-mm optical path quartz cuvette (White Bear Photonics, White Bear Lake, Minnesota, USA), were used. Specifically, the cuvette was rinsed with aqua regia (mixture of 36.5- 38.0 w / w% HCl and 68.0-70.0 w / w% HNO3with a volume ratio of 3:1) and water sequentially and then dried before use. A reference spectrum was acquired with water alone, thus correcting for any optical losses due to intrinsic water absorption and reflection at the air−quartz interfaces.

[0139] For transmission measurements of scattering phantoms, the phantom with the mold was placed in the light path of the UV-Vis spectrometer. A clear hydrogel without silica scatterers in the mold was used as the reference to correct for any attenuation due to baseline absorption and reflection at the air-hydrogel interfaces.

[0140] For transmission measurements of ex vivo tissues, the tissue sample was first mounted on a microscope slide (precleaned, 12-550-15, Fisher Scientific), which was placed in the light path of the UV-Vis spectrometer to ensure that the entire incident beam was blocked by the tissue. For dynamic UV-Vis experiments, the samples were attached to one side of a 1 cm path cuvette holding 3 mL of the dye solution and time-elapsed measurements were carried out with a time interval of 5 minutes until the transmission reached a plateau. Numerical simulation of light propagation in water and scattering phantoms

[0141] The two-dimensional finite difference time domain (FDTD, Lumerical) method was used to simulate the electric (E) and magnetic (H) field of light propagating through an area of 10 μm × 10 μm or 600 μm × 1000 μm that contains two components: silica scatterers and background medium (water or an aqueous solution of tartrazine). A plane wave was launched from the bottom of the simulation box and a tight-focused Gaussian source with a numerical aperture (NA) of 0.25, matching the objective used in imaging, was constructed with 400 plane waves using a thin-lens approximation. A virtual lens with a focal length of 200 μm was placed 100 μm away from the scattering medium to refocus the light, mimicking the formation of an image on a camera. For scattering phantoms, silica scatterers with a diameter of 1 μm were randomly placed in the simulation domain with a density of 0.3 μm−2and 0.003 μm−2. The RIs of water and silica scatterers were taken from the literature, and the RI of the tartrazine solution was taken from ellipsometry measurement (FIGs. 1A-1N). Note that the refractive indices of water and the aqueous solution of tartrazine are the same as their corresponding hydrogels containing 6 mg / mL agarose. The simulation time was set as 7500 fs, with a time stability factor of 0.9. Due to the large simulation domain, mesh accuracy was set at 2 with 10 mesh points per wavelength toATTORNEY DOCKET NO.221910-2720 balance the simulation running time and accuracy, and a conformal variant of 0 was used for mesh setting. The boundary was set as 5 perfectly matching layers. Vertebrate animal subjects

[0142] Adult (20 to 30 g) male and female C57BL / 6J mice (for brain vascular imaging, 14-15 weeks old, Jackson Laboratory, Bar Harbor, Maine, USA), young (10 to 14 g) female BALB / cJ mice and female C57BL / 6J (for muscle sarcomere imaging, 3-4 weeks old, Jackson Laboratory), young (10-15 g) female thy1-YFP-H and C57BL / 6J mice (for brightfield imaging of abdominal organs, 3-4 weeks old, Jackson Laboratory), and adult (15 to 30 g) male and female choline acetyltransferase (ChAT)-Cre-tdTomato mice (for fluorescence imaging of enteric nervous system, 7-12 weeks old) were the animal subjects used in this study. Mice were group-housed on a 12 h: 12 h light:dark cycle in the Stanford University Veterinary Service Center (VSC) with food and water provided ad libitum as appropriate. All animal experiments conducted were approved by Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC) in accordance with Public Health Service Policy on Human Care of Laboratory Animals guidelines and were approved by the Institutional Animal Care and Use Committees of Stanford University. Topical application of dye solutions on live mice

[0143] Mice were weighed and anesthetized via intraperitoneal (i.p.) injection of a mixture of 16 mg / kg ketamine (Dechra Veterinary Products, Overland Park, Kansas, USA) and 0.2 mg / kg dexdomitor (Dexmedesed; Dechra Veterinary Products) via insulin syringes outfitted with a 30G needle. The degree of anesthesia was verified via the toe pinch method. To maintain the body temperature and prevent hypothermia, a homeothermic blanket (Harvard Apparatus, Holliston, Massachusetts, USA) was set to 40 °C and placed underneath the anesthetized mouse. Lubrication eye gel (GenTeal Tears, Alcon, Fort Worth, Texas, USA) was applied on both eyes of the mouse to moisturize the eye surface. Hair removal lotion (Nair, Church & Dwight, Ewing Township, New Jersey, USA) was used for depilation of the mouse abdomen, head, and hindlimbs.

[0144] For topical application, a gel containing the dye molecules was prepared by mixing the low melting temperature agarose with different absorbing molecule solutions (tartrazine sodium salt: 160-320 mg / mL; 4-aminoantipyrine: 600 mg / mL; sunset yellow sodium salt: 390 mg / mL) in a 20 mL scintillation vial (FS74504-20, Fisher Scientific) to reach a final agarose concentration of 3-6 mg mL−1. The mixture was then heated in an oven at 70 °C for at least 10 min, followed by refrigeration at 4 °C for 10 min for solidification. For imaging the gut, brain, and sarcomeres, theATTORNEY DOCKET NO.221910-2720 gel was topically applied to the depilated abdomen, scalp, and the medial part of the hindlimb skin, respectively. The underlying tissues and organs became transparent to the naked eye at ca. 5 min after gentle massaging of the topically applied gel on the skin. Bright field imaging of abdominal organs in live mice

[0145] The hair on the mouse’s abdomen was removed prior to the topical application of the gel containing absorbing molecules. In this study, both tartrazine sodium salt and 4-aminoantipyrine have demonstrated their ability to achieve abdominal transparency. After the abdominal tissue has sufficiently absorbed the dye molecules, the gel is replaced with a clear lubricating eye gel (GenTeal Tears, Alcon, Fort Worth, Texas, USA). Subsequently, the area is covered with a coverslip to prepare it for imaging. The mouse was then placed on a heating pad and exposed to white light illumination (10,000 cd sr m-2) during imaging. Brightfield images were captured using a home-built imaging system equipped with a color CCD camera (DCU224C, Thorlabs). The CCD camera has an array size of 1280 x 1024 and a sensitivity range from 400 nm to 700 nm, where the quantum efficiency is above 50%. The camera was outfitted with a Tamron AF 90mm f / 2.8 Di SP AF / MF 1:1 Macro Lens to capture the mouse abdominal images. No filters were used for imaging. An exposure time of 50 ms was utilized for taking brightfield images. Laser speckle contrast imaging of brain vasculature in live mice

[0146] The depilated mouse head (with or without the application of the gel containing dye molecules) was imaged through a laser speckle imaging system (RWD Life Science Inc., Kent, Delaware, USA) under the sliding mode of imaging with an acquisition time of 5 ms per frame. An incident power density of 6.68 mW / cm2was used for the 785 nm laser. Consecutive frames collected in 15 s were averaged to improve the signal-to-noise ratio of all images. All intensity profiles are normalized between the maximum and minimum values in both curves of each plot. In this study, tartrazine sodium salt, 4-aminoantipyrine, and sunset yellow sodium salt have all demonstrated their ability to achieve abdominal transparency. Second-harmonic generation imaging of hindlimb sarcomeres in live mice

[0147] After topical application of tartrazine-containing gel on the medial part of the hindlimb skin, the sarcomere features in the underlying hindlimb muscles were imaged at a depth of ~220 μm below the surface of the skin in live mice. Specifically, in vivo sarcomere imaging was performed on a laser-scanning microscope (Prairie) equipped with a wavelength-tunable titanium–sapphire laser (Mai Tai, Spectra-Physics).1000 nm or 1040 nm illumination was used with an incident laserATTORNEY DOCKET NO.221910-2720 power of 0.66 mW and the emission was band-pass filtered in the 500 / 10 or 525 / 50 channel, respectively. A microscopic objective (Olympus) with a numerical aperture (NA) of 0.3, 0.5, or 0.8 focused illumination onto the mice limb. Images were acquired at 512 × 512 pixels with 6 μs pixel dwell time and averaged over 64 consecutive frames. A band-pass filter selective for 1–5 μm periods was then applied in ImageJ, following a previously reported protocol for sarcomere imaging. Fluorescence imaging of myenteric plexus in live mice

[0148] After the topical application of the absorbing gel to the abdomen of ChAT-Cre-tdTomato mice, the fluorescence signal from tdTomato was collected using a laser-scanning two-photon fluorescence microscope (Prairie, FIGs. 2B-2C). The imaging was performed under 920 nm illumination with an incident laser power of 46.8 mW, and the emission was band-pass filtered at 595 / 50. An objective with a 0.3 numerical aperture (NA) from Olympus (PlanN) focused the illumination onto the mouse’s abdomen. Images were acquired at a resolution of 512 × 512 pixels with a 6 μs pixel dwell time. Intensity profiles were acquired along the lines indicated in images taken at different depths, with the signal-to-background ratio (SBR) computed as the ratio of the peak intensity to the average baseline signal from the background. For faster imaging speeds (FIG.2D), an epi-fluorescence imaging system (Bergamo imaging platform, Thorlabs) was used. This system was operated under 561 nm illumination, and its emission was band-pass filtered at 607 / 70. A 0.5 NA objective from Thorlabs focused the illumination onto the mouse’s abdomen for imaging. The small-NA objective ensures a larger depth of field, thereby accommodating the motion of the GI tract along the z-axis during dynamic microscopic imaging.

[0149] After the in-vivo imaging experiment, the abdominal wall of the imaged subject was removed, and the tissues were dissected for ex-vivo fluorescence imaging using a Zeiss 980 confocal microscope. For the quantitative comparison of fluorescence intensity, Fiji software was utilized based on methods described in previous publications. Regions of interest (ROIs) measuring 150.81 µm × 150.81 µm were selected on the focal plane for each image. The region exhibiting fluorescence signals was identified using the auto-thresholding function in Fiji, while the remaining portion of the image was designated as the background region. Fiji then separately measured the mean intensity for both the signal and background regions. The fluorescence intensity was subsequently calculated using the formula: fluorescence intensity = mean(signal) – mean(background). Analysis of the dynamic movement of the myenteric plexusATTORNEY DOCKET NO.221910-2720

[0150] A custom Matlab code was developed to process the results from the fluorescence imaging. Initially, out-of-focus images due to global body movements were discarded. Subsequently, displacement vector maps between adjacent frames were computed using the particle image velocimetry (PIV) method. For calculating the displacement at a specific location between two images, a window of 128 × 128 pixels (equivalent to 93.44 × 93.44 μm) was centered around the location of interest in the first image. Simultaneously, a search window of 256 × 256 pixels (equivalent to 186.88 × 186.88 μm) was centered around the same location in the subsequent image. A two-dimensional cross-correlation was computed using Matlab’s built-in function, normxcorr2. The local peak of this cross-correlation, identified by the findpeaks function in Matlab, was designated as the displacement vector. This procedure was iteratively applied to each region of interest throughout the entire field of view, resulting in the two-dimensional displacement map (FIGs.2F-2J). Metabolic study for quantification of excretion of injected tartrazine

[0151] Three mice were individually housed in a metabolic cage after being intramuscularly injected with 50 µL of 0.62 M tartrazine solution prepared with 1x PBS. Feces and urine samples were collected in the following 48 h. Specifically, tartrazine remains the same molecule in the urine, as evidenced by an identical spectrum in the urine, while a metabolized form of tartrazine is found in the feces. The concentration of unmetabolized Dye-4 in the urine was determined based on the absorption maximum at 428 nm, measured by the UV-vis spectrometer. In addition, feces were collected, hand ground, and mixed with DI water to a final concentration of 10 mg mL−1. The solution was then kept rotating at 70 °C for 15 h (micro autoblot hybridization oven, Bellco Glass Inc., Vineland, New Jersey, USA) and filtered through a 0.45 μm syringe filter (SLHVR33RS, Sigma-Aldrich) before the UV-Vis measurement. The metabolites of tartrazine are absorbing in the 300-700 nm range, thus allowing use of an integrated area under the curve to quantify their concentration by assuming the linearity in Beer-Lambert law. Replication

[0152] The sample size for each experiment was determined by power analysis to ensure statistical rigor for all comparisons:ATTORNEY DOCKET NO.221910-2720 where ^^ = 0.05, ^^1−^^ / 2= 1.96, ^^ = 0.20, ^^1−^^= 0.84, ^^0is the mean of the outcome variable for the control (without treatment), ^^1is the mean of pilot test samples after tissue transparency is achieved, and σ is the standard deviation of the outcome variable.

[0153] Brightfield imaging internal organs through the transparent abdominal wall was repeated in 6 mice. Laser speckle contrast imaging of brain vasculature was repeated in 8 mice. SHG imaging of muscle sarcomeres was repeated in 4 mice. Two-photon fluorescence imaging and widefield fluorescence microscope imaging of myenteric plexus was repeated in 4 mice. Statistics

[0154] Comparison between groups was evaluated with a Paired Sample t Test (OriginPro) and one-way analysis of variance (ANOVA). P^<^0.05 was considered statistically significant. Simplification of a multi-oscillator Lorentz model

[0155] When molecules, including absorbing molecules and those of conventional optical clearing agents (OCAs), dissolve in a solvent (e.g., water), each molecule may exhibit multiple resonance absorption peaks. Each of these peaks can be modeled as a harmonic oscillator. Therefore, a solution containing either a single solute or multiple solutes can be treated according to a multi-oscillator Lorentz model. The relative permittivity εrof the solution is the sum over the various oscillators (with a total of N oscillators):frequency ωj, the damping constant γj, and the plasma frequency ωp,j. In addition, ω in Eq. (8) represents the frequency of interest, e.g., the frequency (and its associated wavelength) at which imaging is performed. Furthermore, εmis the relative permittivity of the medium, which represents the effect of all oscillators in the solvent molecules (e.g., water molecules) at higher frequencies.

[0156] For a particular frequency of interest ω (corresponding to an imaging wavelength of λ), which is typically situated in the visible spectrum for imaging purposes, the sum in Eq. (8) is usually dominated by the term with ωjclosest to ω. This is because the term with ωjclosest to ω tends to “explode” when ωj≈ ω. Since all ωj> ω, it follows that within the sum, arranged in descending order of ωj, the term corresponding to the smallest ωj– which is the last in the sequence – will have the largest value. Note that this last term corresponds to the longest-ATTORNEY DOCKET NO.221910-2720 wavelength absorption peak occurring just before the imaging wavelength λ. Thus, this Nthterm can be isolated from the sum, focusing the analysis on its contribution:peaks except for the last one occurring just before the imaging wavelength. With this simplification, Eq. (9) becomesin a single term ε∞ that contains the contributions of all high-frequency oscillators from the solvent and solute molecules except for the oscillator that represents the longest-wavelength absorption peak:the importance of the longest-wavelength absorption peak. Derivation of the molar ∆n' coefficient from the Lorentz model

[0159] Starting from Eq. (2):an ω was a resonance at ω = ω0– Δω. This choice is justified because to achieve optical transparency, it is crucial to avoid operating at the resonance peak, where absorption causes significant attenuation, while still benefiting from the increase in the real refractive index near the resonance. This Δω should have a similarATTORNEY DOCKET NO.221910-2720 magnitude as γ, indicating an approximate location at the base of the absorption peak. Inserting ω = ω0– Δω into Eq. (2) yieldsasa where the absorption is minimal, i.e., B ≈ 0. Thus:ATTORNEY DOCKET NO.221910-2720

[0166] Applying Taylor series expansion yieldswater). Assuming the medium has no absorption at the imaging wavelength, which is true for pure water in the visible spectrum, yieldsthe definition of the plasma frequency:e and m are the charge and mass per electron, and ^^0is vacuum permittivity. Eq. (21) can be expressed using more familiar terms of the concentration as followsconstant, and Z is the number of electrons contributing to the Lorentz oscillators per solute molecule. For absorption in the near-UV (NUV, 300-400 nm) or blue regions of the visible spectrum, delocalized π electrons contribute to these oscillators. Comparing Eq. (22) with the definition of molar ∆n' coefficient in Eq. (6) yieldsATTORNEY DOCKET NO.221910-2720

[0168] Eq. (23) recapitulates key principles discussed in previously, governing the efficacy of an absorbing molecule in raising the n' of the medium it is dissolved in:

[0169] Given the ω0−1scaling, absorbing molecules with low resonance frequencies are more effective in raising the n' of the medium. This explains why absorbing molecules are more efficient in raising the n' of the medium than conventional OCAs with no absorption in the NUV or blue regions of the visible spectrum.

[0170] Given the γ−1scaling, absorbing molecules with a low damping constant γ and thus sharper absorption peaks are more effective in raising the n' of the medium.

[0171] Given the Z scaling, π-conjugated molecules with more delocalized electrons are more effective in raising the n' of the medium. Example 3: Ampyrone for Color-Neutral Optical Transparency

[0172] Ampyrone is a potent UV absorber that enables color-neutral optical transparency across the visible spectrum. An ideal absorbing molecule should have the majority of its absorption bands in the ultraviolet (UV) spectrum, leaving most of the visible spectrum unobstructed for imaging while leveraging RI modulation in the visible range, enabled by intense UV absorption. This design can exploit the Kramers-Kronig relations, which describe the causal relationship between the wavelength-dependent variation of the real (n) and imaginary (k) parts of a material’s complex refractive index. These relationships are expressed analytically as follows by Equation 1. The imaginary RI, ^^^^^^, is related to the absorption coefficient ^^^^^^ by ^^^^^^ ൌఈ^ఒ^⋅ఒ ସగ (24)

[0173] Based on this theoreticalintegrated absorption below the wavelength of interest while minimizing absorption at the wavelength of interest. This requirement suggests a sharp absorption edge at a wavelength immediately below the desired imaging wavelengths (FIGs. 12A-12D). Guided by this design principle, phenazone, also known as antipyrine, was first explored. Phenazone has been previously reported as an ex vivo RI-matching agent, due to its high refractive index and low visible absorption. However, effective phenazone solutions typically require concentrations of 55% or higher, resulting in a mixture where phenazone dominates and water molecules constitute less than half of the solution.ATTORNEY DOCKET NO.221910-2720

[0174] We sought to understand the relative inefficiency of phenazone by measuring its UV-Vis transmission spectrum in an aqueous solution. The absorption spectrum of phenazone in the UV- visible range reveals intense absorption below 300 nm, with a rapid decay to minimal absorption beyond 320 nm (FIG.5A). For fluorescence imaging with GFP and more red-shifted fluorophores, there is a significant gap of at least 160 nm (from the sharp absorption edge at 320 nm to GFP’s excitation wavelength at 488 nm) that does not contribute to RI increase in the visible spectrum. To address this inefficiency, strategies aimed at red-shifting the absorption edge to fill this gap in the absorption spectrum would be an effective approach for identifying more efficient RI-matching agents that function at lower and more physiologically tolerable concentrations.

[0175] To red-shift the absorption edge, an empirical principle well known to organic chemists for synthesizing donor-acceptor colorants was applied. Specifically, it is established that electron- donating groups, particularly amine groups, conjugated with an extended chromophore system can induce both bathochromic (red-shifting) and hyperchromic (up-shifting) shifts in the absorption peak. This effect arises from the extension of the π system through the involvement of nonbonding p-orbitals and the intensification of the molecular dipole. Specifically, it was hypothesized that an amine group addition at the C4 site of the central pyrazole backbone of phenazone can significantly red-shift the absorption edge, thus bringing the resonance closer to the visible wavelengths. It was also hypothesized that this amine group can create a push-pull electron donor-acceptor system, which is also responsible for the redshift. The UV-Vis transmission spectrum of the resulting molecule, ampyrone, exhibits a red-shifted absorption edge extending to approximately 400 nm (FIG.5A), significantly increasing the total absorption area. As a result, ampyrone appears pale yellow (FIGs.13A-13B) while otherwise remaining largely color-neutral across the visible spectrum.

[0176] The intense UV absorption of ampyrone leads to efficient RI modulation with minimal absorption across the visible spectrum. Ampyrone exhibits a broad absorption peak at 220-320 nm (FIG.13A), with shoulder peak absorption extending well beyond 400 nm (FIG.5A and 13C). As a result, after dissolving in water, ampyrone can significantly increase the real RI, n, through the Kramers-Kronig relations (FIG.13D). Specifically, the RI modulation of water as a function of ampyrone concentration (% w / w) and wavelength (nm) (FIG. 5B) reveals that peak index modulation occurs at ~310 nm, after which it rapidly plateaus across the visible and near-infrared wavelengths. It was further examined how n varies with concentration at multiple selected wavelengths (FIG.5C). Near the RI maximum at 300 nm, within the high-dispersion regime, n increases more rapidly with concentration. However, moving into the visible spectrum (400–800ATTORNEY DOCKET NO.221910-2720 nm), the dependence of n on concentration remains relatively stable, reinforcing the plateau behavior.

[0177] To further corroborate ampyrone’s intense absorption in the UV spectrum and minimal absorption across the visible spectrum, the imaginary refractive index (k) was plotted as a function of both concentration and wavelength (FIG.5D). This analysis reveals several key findings. First, ampyrone exhibits the most intense UV absorption (e.g., at 310 nm) at equivalent concentrations compared to commonly used index-matching agents (FIG. 5E). This exceptionally strong UV absorption—far exceeding that of other index-matching agents—underpins its RI modulation efficiency in the visible spectrum (FIGs.5B-5C). Second, beyond the UV spectrum, ampyrone demonstrates minimal absorption across the entire visible range (FIG.5D), enabling imaging with common fluorescent proteins while minimizing attenuation in the transparency window. When exposed to air at room temperature, ampyrone gradually develops an orange hue, corresponding to an increasing absorption peak at 400 nm (FIGs.13B-13C). However, this additional coloration does not appear to significantly alter the refractive index or chemical composition of ampyrone (FIGs.13D-13E). Third, as a result of its intense UV absorption and negligible visible absorption, ampyrone achieves the most efficient modulation of the real refractive index (n) in aqueous solutions, compared to previously reported in vivo index-matching agents (FIG.5F). These agents include sucrose, glycerol, and dextran (40 kDa) with their full real and imaginary RI spectra as a function of concentration provided in FIGs.14A-14H. Lastly, ampyrone’s small molecular weight (203 g / mol) provides significant advantages in water solubility and diffusion efficiency in tissue.

[0178] Ampyrone achieves transparency in scattering phantoms. To further evaluate the optical performance of ampyrone in enhancing tissue transparency, 5-mm-thick scattering phantoms composed of 1% w / w agarose hydrogel with 1 μm silica particles dispersed within were prepared (FIG.6A). These phantoms were backlit with a grid pattern using an LED panel to assess light transmission at varying ampyrone concentrations. Without ampyrone, the grid lines were completely obscured due to strong light scattering through the phantom. As the ampyrone concentration increased, the grid lines became progressively more visible, with maximum clarity observed at 38% w / w (corresponding to n ~1.43, closely matching the RI of silica particles). However, at 44% w / w, where the RI exceeded the optimal matching condition, the clarity of the grid pattern decreased. This decreased clarity supports the conclusion that the observed transparency arises from the RI modulation of the aqueous component in the scattering phantom by ampyrone.ATTORNEY DOCKET NO.221910-2720

[0179] To systematically quantify the relationship between ampyrone concentration and transparency performance in the silica phantom, phantoms with varying ampyrone concentrations were prepared and, further, the transmitted image of a 1951 USAF resolution test target through 2 mm thick phantoms was imaged (FIGs.6B-6C). At 0% w / w (without ampyrone), the USAF target is almost completely obscured by scattering, with only large features visible beyond 785 nm in the near-infrared (NIR) spectrum. As the ampyrone concentration increases, a progressive reduction in scattering and improved image formation was observed across the visible spectrum. At 17% w / w—an undermatched condition—scattering is already noticeably reduced. By 38% w / w, the USAF targets appear nearly identical to the control target without any scattering phantom.

[0180] To quantify the resolution improvements due to index matching, the modulation transfer function (MTF) was calculated for each image in FIGs. 6B-6C. Although the MTF is more rigorously obtained by taking the Fourier transform of the point spread function (PSF), the USAF target provides a reasonable approximation at specific, discrete spatial frequencies. The MTF for all concentrations at a given wavelength is plotted, demonstrating that 38% w / w ampyrone is optimal for increasing the MTF of the scattering phantom across both the visible and NIR spectra. Even at lower concentrations, there is a significant improvement in MTF compared to the original scattering phantom, suggesting that if perfect RI matching is not required, lower ampyrone concentrations can still yield an observable transparency effect.

[0181] Ampyrone achieves optical transparency in ex vivo skin. Ampyrone’s ability to enhance optical transparency in biological tissue ex vivo was next evaluated. To do this, abdominal skin was harvested from 4-month-old mice and incubated in ampyrone solutions of varying concentrations to assess its ability to induce optical transparency. While 17% w / w ampyrone had minimal effect, partial transparency was observed at 29% w / w, and nearly complete transparency was achieved at 38% w / w. However, higher concentrations, such as 44% w / w, resulted in poor transparency (FIG.7A).

[0182] Transmission spectra analysis showed that skin treated with 38% w / w ampyrone reached up to 30% transmission across the visible spectrum, a substantial increase from the <1% transmission of untreated skin (FIG.7B). Quantitative analysis revealed a >50-fold enhancement in transmission for 38% w / w ampyrone compared to untreated samples (FIG. 7C). Finally, analysis of skin area before and after treatment showed negligible changes across all tested concentrations, indicating that tissue size was preserved after the tissue became transparent (FIG.7D).ATTORNEY DOCKET NO.221910-2720

[0183] Ampyrone achieves optical transparency in the live mouse abdomen. Next, it was aimed to demonstrate the transparency effect in live mice using a 38% w / w ampyrone solution, which had shown maximum transparency in scattering phantoms and ex vivo skin. Although a mouse’s depilated abdominal skin typically appears opaque (FIG. 8A), topical application of 38% w / w ampyrone significantly increased transparency, allowing detailed visualization of internal organs such as the liver and intestines within the abdominal cavity (FIG.8B). It was confirmed that these emerging features originated from internal organs by physically removing the abdominal skin, which revealed similar organ structures (FIG.8C).

[0184] Next, it was aimed to demonstrate the reversibility of the ampyrone treatment process (FIG.8D). After creating a transparent window in the abdominal skin using ampyrone, a saline solution was topically applied to extract the ampyrone molecules, effectively reversing the transparency effect and restoring the skin’s original appearance. Following the reversal, a bioadhesive hydrogel was applied to maintain skin hydration before the mouse was recovered from anesthesia. The animal exhibited normal activity and behavior in the following days, with the skin appearing normal over the next seven days. Hair regrowth was observed in the treated abdominal area thereafter.

[0185] To assess the biosafety of topically applied ampyrone, blood chemistry and hematology assays were conducted on mice that received abdominal treatment with ampyrone. While ampyrone functions as an anti-inflammatory, analgesic, and antipyretic agent, it is not entirely biorthogonal as an index-matching agent. The assay results (FIGs.15A-15P and Table 4) showed no statistically significant differences between saline-treated and ampyrone-treated mice at both 1 day and 14 days post-treatment. Histological analysis of skin tissue from mice treated with saline or ampyrone at these time points revealed no significant differences in tissue morphology or inflammatory response (FIGs.16A-16H and Table 5). Table 4: P-values comparing control and experimental conditions for FIGs.15A-15P onATTORNEY DOCKET NO.221910-2720 HCT 0.31 0.06 ornt. tal ms V: inTable 5: Mean Skin Histology Rankings ar arATTORNEY DOCKET NO.221910-2720 Day 14 27±5 1.3±0.6 1±0 1±0 0.7±0.6 0±0 Ampyrone 0- o th

[0186] We attrbute ampyrones mnma toxcty to t e ow dose and transent nature o the treatment. Specifically, it is estimated that 20 µL of a 38% w / w ampyrone solution penetrated the skin to achieve transparency, while the subsequent reversal step extracted 80% of the skin- absorbed molecules. As a result, only a 0.05–0.17 g / kg body weight dose was effectively administered across all tested animals, significantly lower than the reported LD50 for intraperitoneal ampyrone administration (1.2 g / kg body weight in rodents). This reference on LD50 of ampyrone also highlights ampyrone’s low toxicity as a key factor in its use as a safer analgesic and antipyretic alternative to its parent compound, aminopyrine.

[0187] Ampyrone enables longitudinal two-photon microscopy through the scalp in live mice. Following these demonstrations, it was sought to evaluate ampyrone’s potential to enable deep- tissue imaging across the entire visible spectrum, leveraging its color neutrality. Structural imaging of YFP-expressing neurons was first performed in the cortex of live Thy1-YFP-H mice through the transparent scalp using two-photon excitation fluorescence microscopy. YFP imaging was not compatible with a previous in vivo transparency approach due to the absorption profile of tartrazine, which significantly attenuated YFP fluorescence.

[0188] Traditionally, two-photon fluorescence microscopy of the mouse cortex requires scalp removal and the installation of a cranial window to replace part of the skull, as these overlying tissues cause significant scattering that prevents microscopic imaging of underlying neurons. This limitation is evident in a three-dimensional (3D) reconstruction of two-photon fluorescence (excitation: 920 nm at 110 mW; emission: 525 / 50 nm; field of view per frame: 598 μm x 598 μm; pixel size within frame: 1.17 μm; average per frame: 1; z step size: 5 μm; total acquisition time: 55 s) collected to a depth of 650 μm below the depilated scalp surface, where the only observable autofluorescence signal arises from hair follicles within the most superficial 100 μm of the scalp (FIGs.9A and 17A-17B). In contrast, after treating the scalp with 38% w / w ampyrone to achieveATTORNEY DOCKET NO.221910-2720 transparency, 3D-reconstructed two-photon fluorescence images using the same imaging parameters revealed extensive Thy1-YFP-H neuronal structures extending up to 650 μm below the scalp surface (FIG.9B). In these images, the scalp, skull, and cortex are distinctly delineated based on their fluorescence properties: hair follicle autofluorescence marks the scalp, an absence of signal (dark layer) represents the skull, and YFP signals highlight apical dendrites from pyramidal neurons in the cortex. The presence of hair follicles has minimal impact on the resolution of the smallest apical dendrite structures (FIGs.18A-18C); however, their presence has contributed to a 4x reduction in the two-photon excited fluorescence intensity due to absorption by the pigments in hair follicles in the cleared scalp (FIG.19D).

[0189] Cross-sectional fluorescence images at representative depths of 300 μm and 400 μm below the surface of the scalp further illustrate the contrast between the same mouse before and after achieving scalp transparency (FIGs.9C-9F). Specifically, in the untreated condition, virtually no neuronal signals were detectable through the opaque scalp (FIGs. 9C-9D), whereas after ampyrone treatment, clear dendritic structures became visible at these depths (FIGs.9E-9F). The smallest resolvable dendritic features exhibit widths of approximately 2^μm, with minimal optical aberration (<2× broadening) at this depth, as characterized using standardized point sources (see FIGs. 19A-19C). Comparative imaging of the brain in the same live mouse – through the transparent scalp and skull versus through a cranial window – further confirmed consistent measurements of apical dendrite thickness in cortical pyramidal neurons (FIGs. 18A-18C). Notably, ampyrone induces scalp transparency without penetrating the skull. In mice within the P15-28 age range, scalp transparency alone was sufficient to enable visualization of cortical neuron structures through both the scalp and skull. This is attributed to the fact that, at this age, the scalp is the primary source of light scattering, while the skull remains transparent enough to permit optical transmission. In contrast, mice outside the P15-28 age range exhibit unfavorable optical properties of the skull that prevent microscopic imaging of the brain, even when the scalp is made transparent. This demonstration represents the first successful imaging of YFP- expressing neurons through both the scalp and skull in juvenile mice.

[0190] The transient and reversible scalp transparency enabled repeated imaging of the same neuronal structures in the same animal for longitudinal studies. The rapid changes in brain size during early development pose challenges for conventional intravital window installations. Ampyrone was used to create a transparent scalp window repeatedly, allowing cortical neuron imaging (excitation: 920 nm at 110 mW; emission: 525 / 50 nm for YFP and 460 / 50 for second harmonic generation; field of view per frame: 1.170 mm x 1.170 mm; pixel size within frame: 2.29ATTORNEY DOCKET NO.221910-2720 μm; average per frame: 1; z step size: 5 μm; total acquisition time: 63 s) in the same Thy1-YFP- H mouse over four consecutive days, from postnatal day 22 (P22) to P25 (FIGs.9G-9N). Single cross-sectional images taken at a fixed depth of 250 μm in layer 1 of the primary visual cortex (V1) from the scalp surface on different postnatal days revealed consistent neuronal structures in the same brain region, as evidenced by identical blood vessel features (appearing as negative contrast) across all images. This study demonstrates the feasibility of longitudinal imaging with repeatable scalp transparency in vivo, which could be particularly valuable for studying neurodevelopment in mice.

[0191] Ampyrone enables functional calcium imaging through the scalp in live mice. After successfully demonstrating structural imaging of neurons in the brain through the transparent scalp, it was next aimed to evaluate functional calcium imaging using GCaMP in awake mice. Specifically, injected wild-type mice were transcranially injected with a GCaMP8m adeno- associated virus (AAV) in the right cortical hemisphere on the day of birth (P0). Imaging was performed 21 days postnatally (P21). Similar to FIG.9A, in the presence of an intact scalp, two- photon fluorescence microscopy (excitation: 920 nm at 110 mW; emission: 525 / 50 nm; field of view per frame: 598 μm x 598 μm; pixel size within frame: 1.17 μm; average per frame: 1; z step size: 5 μm; total acquisition time: 16 s) primarily revealed autofluorescence from hair follicles, with minimal discernible cortical features underneath (FIG.10A). In contrast, after rendering the scalp transparent with ampyrone, two-photon microscopy using the same imaging parameters clearly resolved GCaMP8m-labeled neuronal cell bodies in the cortex at a depth of approximately 200 μm from the surface of the scalp (FIG.10B). Cross-sectional fluorescence images at a fixed depth of 170 μm from the surface of the scalp further demonstrated this contrast, with significantly improved visibility of GCaMP8m-labeled cortical features through the transparent scalp (FIGs. 10C-10D).

[0192] We next conducted time-lapse two-photon fluorescence microscopy of GCaMP6f through the transparent scalp in awake behaving head-fixed mice (excitation: 920 nm at 30 mW; emission: 525 / 50 nm; field of view: 1.170 mm x 1.170 mm; pixel size: 4.57 μm; average per frame: 1; acquisition time per frame: 0.81 s). The resulting frame rate – just slightly above 1 fps – is a suboptimal aspect of this imaging experiment. This long acquisition time was attributed to lingering absorption by the unremoved scalp and skull, which attenuates photons emitted by GCaMP6f. Nonetheless, representative time-dependent traces from six selected GCaMP6f- labeled neurons (FIG. 10E) had adequate temporal resolution to resolve distinct calcium transients in response to air-puff stimulation applied to the whiskers and face. Notably, three ofATTORNEY DOCKET NO.221910-2720 the six neurons (neurons 1, 2, and 3) exhibited a clear stimulus response, while the remaining three (neurons 4, 5, and 6) showed minimal response (FIG. 10F). Importantly, the distinct responses of individual neurons ruled out motion artifacts as a source of calcium transients (FIGs. 20A-20I). Fourier transform analysis of the same brain region, imaged both through the transparent scalp and after scalp removal, also hints at the comparable imaging resolution through the transparent scalp to that achieved with the scalp entirely removed (FIGs.21A-21I). These experiments demonstrate that ampyrone enables functional calcium imaging of cortical neurons through the transparent scalp and skull, expanding the potential for noninvasive in vivo neuroimaging in awake and behaving mice.

[0193] Proteomic analysis reveals that ampyrone does not significantly increase apoptotic markers. To further evaluate the biosafety of ampyrone-induced in vivo tissue transparency, proteomic analysis was performed on mice treated with ampyrone on their abdominal skin. Apoptotic markers, particularly members of the caspase and Bcl-2 family, were evaluated, as these proteins play key roles in programmed cell death. The caspase family of cysteine proteases, including Casp3, Casp6, Casp7, Casp8, and Casp9, are predominantly linked to their ability to enact or enable functions related to apoptosis, while Bax, a proapoptotic member of the Bcl-2 family, serves as an established indicator of apoptotic signaling.

[0194] Both male and female mice were treated with ampyrone and allowed to recover for approximately 72 h before protein extraction from the abdominal skin, followed by sample preparation for mass spectrometry (FIG.11A). Control mice underwent an identical procedure with PBS in place of ampyrone. Using a significance threshold of P < 0.05 for fold change enrichment, no significant upregulation of apoptotic markers was observed in ampyrone-treated samples compared to controls (FIGs.11B-11C). These findings suggest that ampyrone does not induce apoptotic signaling in treated tissues at 72 h after achieving and reversing transparency, reinforcing its potential suitability for in vivo tissue transparency applications.

[0195] Discussion. In this study, it was demonstrated that in vivo optical tissue transparency, previously limited to red visible wavelengths in previous work, can now be extended across the entire visible spectrum. This advancement significantly broadens the applicability of the in vivo transparency approach, making it compatible with a wide range of fluorescent proteins and other fluorescent probes throughout the visible spectrum. Leveraging this development, both longitudinal and functional imaging of neurons were successfully performed in the mouse cortex using YFP and GCaMP, respectively, through the intact scalp and skull of live mice.ATTORNEY DOCKET NO.221910-2720

[0196] While previous studies have achieved two-photon fluorescence microscopy through the skull and even more advanced three-photon transcranial imaging, this study is the first demonstration of cellular-resolution imaging of neuronal structures and calcium activity through both the scalp and skull. Although the present work has focused on yellow and green fluorescent proteins, red fluorescent proteins such as mCherry, tdTomato, and RCaMP should also be fully compatible, enabling multiplexed brain imaging with multiple fluorophores in distinct color channels through ampyrone-treated transparent tissue.

[0197] Beyond expanding optical transparency to the full visible spectrum in living tissue, the reversibility of the present transparency approach in the mouse abdominal skin has also been demonstrated, and it has been shown that scalp transparency can be achieved while the mouse remains awake and behaving. This reversible and transient nature of ampyrone treatment ensures biosafety, as supported by blood chemistry, hematology (FIGs.15A-15P), skin histology (FIGs. 16A-16H), and proteomics analyses (FIGs. 11A-11C). Additionally, it enables chronic imaging by allowing the same skin region to be rendered transparent repeatedly over multiple days (FIGs.9A-9N).

[0198] Several limitations exist for this approach in its current form. A key limitation arises from the inherent heterogeneity of RI distribution in biological tissues, which is more complex than that of the silica phantoms. Consequently, matching only a subset of RIs does not achieve the same degree of transparency. Therefore, the transparency achieved in ex vivo and in vivo tissues reflects reduced – but not fully eliminated – scattering. In addition, although this study demonstrates excellent recovery following transient ampyrone treatment, the use of a high concentration of ampyrone – at 38% w / w – requires thorough evaluation of its long-term effects in mice before progressing to larger animal models. Furthermore, achieving optical transparency in internal organs presents greater challenges due to the necessity of more invasive delivery methods and the increased vascularization, which may lead to rapid clearance of RI-matching agents. Lastly, cell proliferation assays should be performed if ampyrone is used to achieve optical transparency in 2D / 3D cell and organoid cultures, or for in vivo applications involving systemic delivery.

[0199] Future advancements in absorbing molecule design could further enhance this approach. Fluorophores with stronger ultraviolet resonances and broader absorption spectra could improve transparency efficiency, allowing for even lower concentrations to achieve effective RI matching in tissue. Alternatively, engineered dyes with narrow absorption profiles could be optimized forATTORNEY DOCKET NO.221910-2720 operation near specific resonances, enhancing the precision and efficacy of optical tissue transparency. Example 4: Materials and Methods

[0200] Chemicals and materials. Ampyrone was purchased from Sigma-Aldrich (33528-100G-R, batch BCCK4073, Sigma-Aldrich, Burlington, Massachusetts, USA) and stored in an MBRAUN Unilab nitrogen purged glove box fridge (14-091, MBRAUN, Garching, Germany) at -20°C before use. Powder aliquots were stored under vacuum at room temperature for <1 week before immediate use and were covered in foil to prevent exposure to ambient light.

[0201] For ex vivo experiments, ampyrone solutions were prepared by dissolving 200 mg, 400 mg, 600 mg, or 800 mg of ampyrone in 1 mL of deionized water at 40°C for 10 min, resulting in concentrations of 17% w / w, 29% w / w, 38% w / w, and 44% w / w, respectively. Occasional shaking was used to ensure complete dissolution. After preparation, ampyrone solutions were stored at ambient conditions and used within 12 h; any unused solution was discarded to maintain experimental consistency.

[0202] For in vivo experiments, ampyrone solutions were prepared by dissolving 600 mg of ampyrone in 1 mL of deionized water at 40°C for 10 min to achieve a 38% w / w concentration. Occasional shaking was used to ensure complete dissolution. The solutions were stored at ambient conditions and used within 12 h; any remaining solution was discarded to maintain experimental consistency.

[0203] Phenazone (A5882-100G, Sigma-Aldrich, Burlington, Massachusetts, USA), glycerol (G5516-1L, Sigma-Aldrich, Burlington, Massachusetts, USA), and dextran (MW: 40 kDa, 31389- 25G, Sigma-Aldrich, Burlington, Massachusetts, USA) were all purchased from Sigma-Aldrich. Sucrose (S5-500, Fisher Scientific Company, Pittsburgh, Pennsylvania, USA) was purchased from Fisher Scientific Company. Deionized water was obtained from 18.2 MOhm deionized Milli- Q Integral 10 (7003, EMD Millipore, Burlington, Massachusetts, USA) and used as the solvent for all experiments.

[0204] Spectroscopic ellipsometry. All spectroscopic ellipsometry experiments were performed on a Horiba Jobin Yvon UVISEL (2015 model, Horiba Jobin Yvon IBH Limited, Irvine, California, USA) spectroscopic ellipsometer. The bulk complex refractive index was measured for each solution with a volume of 2.5 mL. Tissue-Tek Cryomolds (4557, Sakura Finetek, 25 mm x 20 mm x 5 mm, Torrance, California, USA) were used to hold the solution, secured to the UVISEL stageATTORNEY DOCKET NO.221910-2720 with a built-in vacuum chuck. To eliminate reflections from surfaces other than the top liquid surface of the sample, 320-grit sandpaper (SiC A-99, Partsmaster 881-5-0320, Dallas, Texas, USA) was attached to the bottom of the cryomold using double-sided tape (3M 34-8724-5691-7, Saint Paul, Minnesota, USA). This ensured that only the bulk complex refractive index of the liquid sample was measured, without the need for multilayer postprocessing of the raw ellipsometric data. Spectra were taken from 250 nm to 850 nm with a 2 nm step size and a 200 ms dwell time.

[0205] UV-Visible spectrophotometry. All absorption and transmission experiments were performed on a Thermo-Scientific Evolution 350 UV-Visible spectrophotometer (Thermo- Scientific, Waltham, Massachusetts, USA). Fused quartz cuvettes with 1 mm pathlength (Aireka Scientific Co Ltd, Hong Kong, China) were used for all liquid measurements. The UV-Visible transmission spectra of ampyrone, phenazone, and tartrazine were measured at concentrations of 100 mg / mL for each compound in FIG.5A. For transmission measurements of mouse skin samples, the samples were placed on glass slides (3” x 1” x 1 mm, 12-544-1, Fisher Scientific, Pittsburgh, PA, USA) and secured with double-sided tape (34-8724-5691-7, 3M, Saint Paul, MN, USA) to built-in cuvette holders (Standard Cell Holder for Thermo 350, Thermo-Scientific, Waltham, MA, USA) within the UV-Vis spectrophotometer. The double-sided tape was placed around the optical window of the cuvette holder to avoid interfering with the spectrometry measurements. Max Bond Super Glue (Krazy Glue, High Point, North Carolina, USA) was used to glue the corners of the skin if the tissue failed to adhere. Spectra were taken from 190 nm to 1100 nm with 0.25 s dwell time, 1 nm step size, and 4 nm bandwidth.

[0206] Scattering phantom imaging. For phantom images in FIG. 6A, scattering phantoms measuring 15 mm × 15 mm × 5 mm were prepared by adding 0, 400, 800, 1200, or 1600 mg of ampyrone (corresponding to % w / w concentrations of 0%, 17%, 29%, 38%, and 44%, respectively) along with 20 mg of SeaPlaque agarose (50101, Lonza, Rockland, MO, USA) into a 20 mL scintillation vial (DWK986546, Sigma-Aldrich, Burlington, MA, USA).2 mL of 10 mg / mL 1 μm silica particle suspension (SISN1000, nanoComposix, San Diego, California, USA) was then added to each vial. The suspension was heated at 80°C for 10 min and then thoroughly mixed using a vortex mixer. It was then heated for another 10 min at 80°C and mixed again with a vortex mixer. Next, 1.2 mL of each suspension was cast into Tissue-Tek Cryomolds (15 mm × 15 mm × 5 mm, 4557, Sakura Finetek, Torrance, CA, USA) and sealed with a coverslip to ensure a phantom thickness of exactly 5 mm. The samples were chilled on ice for 10 min to set the gel before imaging. The samples were placed on a 300 lumen LED light board (A4 LED Light Board, Comzler, Dongguan, Guangdong, China) with an attached 1 x 1 mm grid pattern printed on aATTORNEY DOCKET NO.221910-2720 transparency film (Gwybkq Transparency Film, 8.5 x 11 Inches, Amazon, Seattle, WA, USA). Imaging was done with a Canon EOS Rebel T6 Digital SLR camera (DS126621, Canon, Tokyo, Japan) equipped with a Canon EFS 18-55 mm MACRO 0.25 m / 0.8 ft lens (226064565741, Canon, Tokyo, Japan).

[0207] USAF resolution target imaging. For USAF resolution target imaging, circularly shaped scattering phantoms were used. Specifically, scattering phantoms with a 2 mm thickness and a 6 mm diameter were prepared by stacking two CultureWell reusable gaskets (CW-8R-1.0, Grace Bio-Labs, Bend, Oregon, USA) onto glass slides (Fisherbrand premium plain glass microscope slides, 3 in x 1 in x 1 mm, 12-544-1, Fisher Scientific, Pittsburgh, Pennsylvania, USA). Scattering phantoms were prepared by adding 0, 20, 40, 60, or 80 mg of ampyrone (corresponding to concentrations in % w / w of 0%, 17%, 29%, 38%, and 44%, respectively) with 1 mg of SeaPlaque agarose (50101, Lonza, Rockland, Missouri, USA) to a 1.8 mL scintillation vial (03338AA, Fisher Scientific, Pittsburgh, PA, USA).100 μL of 10 mg / mL 1 μm silica particle suspension (SISN1000, nanoComposix, San Diego, California, USA) was then added to each vial. The suspension was heated at 80°C for 10 min and then thoroughly mixed using a vortex mixer. It was then heated for another 10 min at 80°C and mixed again with a vortex mixer. The suspension was then cast into the silicon gaskets. Each gasket can hold 8 separate wells, with each well containing a different sample with a different ampyrone concentration. Another glass slide was placed on top to seal the samples in each of the gaskets. The samples in the gaskets were chilled on ice for 10 min to set the gel before imaging.

[0208] A negative 1951 USAF resolution test target (R3L3S1N, Throlabs, Newton, New Jersey, USA) was secured to the stage of a Leica DM2700M microscope (Leica, Wetzler, Germany) with a 10X (0.25 BD N PLAN EPI) objective (Leica, Wetzler, Germany). The USAF target and scattering phantom sample being imaged were centered in the field of view of the microscope. The target was secured with tape to prevent movement while the gaskets were moved to center different wells with different phantom samples. Images were acquired with a Hamamatsu C11440- 22CU digital camera (Hamamatsu, Shizuoka, Japan). For FIG.6C, these images were digitally cropped such that only the group 6-7 elements (the two groups with the smallest and second smallest features) of the USAF target were visible. The following filters were used: 450 / 10 (FBH450-10, Thorlabs, Newton, New Jersey, USA), 525 / 45 (BrightLine Fluorescence Filter 525 / 45, Semrock, Northbrook, Illinois, USA), 600 / 10 (FB600-10, Thorlabs, Newton, New Jersey, USA), 680 / 10 (FB680-10, Thorlabs, Newton, New Jersey, USA), and 785LP (Edgebasic Long Wave Pass 785, Semrock, Northbrook, Illinois, USA). To quantify the contrast of the USAF targetATTORNEY DOCKET NO.221910-2720 through the scattering phantoms, the modulation transfer function (MTF) was calculated as described below in the “Modulation transfer function (MTF) calculation” section.

[0209] Modulation transfer function (MTF) calculation. The MTF is one common metric for quantifying the contrast of an optical system at specific spatial frequencies. It quantifies the ability of a system to capture detailed structures in an image and is based on the spatial frequency of periodic patterns such as line or sine waves. These features are measured in units of line pairs per millimeter (lp mm-1). The 1951 USAF test target is one of the most popular image targets for quantifying the MTF, although it is limited by its discrete, rather than continuous, spatial frequency support. The MTF itself can be calculated by first extracting the normalized difference between the maximum and minimum transmitted light intensities at a specific spatial frequency from an image of a USAF test target. The MTF can then be calculated as: ^^^^^^ ൌூ^ೌ^ିூ^^^ூ^ೌ^ାூ^^^(25) where Imaxis the maximum intensity of aand Iminis the minimum intensity of the same feature.

[0210] Vertebrate animal subjects. Mice were group-housed on a 12 h:12 h light:dark cycle in the Stanford University Veterinary Service Center (VSC) with food and water provided ad libitum. All animal experiments conducted were approved by Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC) in accordance with Public Health Service Policy on Humane Care of Laboratory Animals guidelines and were approved by the Institutional Animal Care and Use Committees of Stanford University. All animal experimental procedures, including mouse tissue harvesting, in vivo abdominal imaging, two-photon fluorescence imaging of the live mouse brain, protein extraction and mass spectrometry from mouse skin, and histopathological analysis of mouse tissues, are detailed in the “Supplementary Materials and Methods” section of the Supporting Information.

[0211] Replication. The sample size for each experiment was determined by power analysis to ensure statistical rigor for all comparisons. Sample size, n, was calculated as: ଶ ^^ ൌ ^^భష^ / మା^భష^^^^ (26)where ^^=0.05, ^^^ିఈ / ଶ=1.96,theexperimental condition, ^^^is the mean of the outcome variable for the control, and ^^ is the standard deviation of the outcome variable.ATTORNEY DOCKET NO.221910-2720

[0212] Statistics. Comparison between groups was evaluated with a two-tailed, heteroscedastic, t-test. P<0.05 was considered statistically significant. Example 5: Optical Transparency in Mouse Skin and In Vivo

[0213] Achieving optical transparency in ex vivo mouse skin. C57BL / 6J mice (male and female, 8-10 weeks old, 20-30 g) from Jackson Labs (Sacramento, California, USA) were used for demonstrating optical transparency in ex vivo mouse skin tissue. The mice were euthanized using carbon dioxide via inhalation, in accordance with the APLAC protocol, followed by cervical dislocation to ensure death. The abdominal skin was then surgically removed after depilation by incising an approximately 10 mm x 20 mm rectangle of abdominal skin and peritoneum from the center of the abdomen. The peritoneum was subsequently removed from the skin. One mouse was used for a single skin sample. Skin samples were glued by the corners onto glass coverslips with Max Bond Super Glue (Krazy Glue, High Point, North Carolina, USA) and placed in a 12 well plate, with one sample per well. The 12 well plate was placed on a 300 lumen LED light board (A4 LED Light Board, Comzler, Dongguan, Guangdong, China) with an attached 1 x 1 mm grid pattern printed on transparency film (Gwybkq Transparency Film, 8.5 x 11 Inches, Amazon, Seattle, WA, USA) in an orbital shaker (MaxQ 4000, Thermo Scientific, Waltham, Massachusetts, USA) set to 40°C. Samples were soaked in aqueous solutions of ampyrone at concentrations of 17%, 29%, 38%, or 44% w / w for 24 h at 40°C without shaking. Images before and after soaking were taken with a digital camera (DCU223M, ThorLabs, Newton, New Jersey, USA) using a Xenon variable aperture f / 0.9525 mm focal length lens (Model 13238999, Schneider-Kreuznach, Bad Kreuznach, Germany) with 200 ms exposure time and f / 11 aperture. For obtaining the T% value for each sample, a blank coverslip identical to the ones used to hold the abdominal skin was used as the baseline (i.e., T% = 100%) before each measurement. The blank coverslip was attached to the cuvette holder. For obtaining the T / Toriginalvalue for each sample, the T% value for the sample after soaking was divided by the T% value for the same sample before soaking.

[0214] Achieving optical transparency for in vivo abdominal imaging. C57BL / 6J and BALB / cJ mice (male and female, 3-4 weeks old, 9-15 g) from Jackson Labs (Sacramento, California, USA) were used for in vivo abdominal imaging. Note that mice older than 4 weeks and weighing more than 15 g can also develop transparent abdominal skin when treated with ampyrone as described below. However, because these older mice typically begin to accumulate adipose tissue beneath the abdominal skin, the abdominal organs remain obscured by the still-opaque fat, even though the skin itself becomes significantly more transparent. This has been confirmed by a recent reportATTORNEY DOCKET NO.221910-2720 of achieving transparency in the abdominal skin of mice weighing more than 15 g with ampyrone. Therefore, for the purpose of direct, naked-eye visualization of abdominal organs, it is recommended to use mice younger than 4 weeks and weighing less than 15 g.

[0215] The mice were anesthetized (Animal Anesthesia Vaporizer, RWD, Sugar Land, Texas, USA) with IsospireTMisoflurane (Dechra Veterinary Products, Overland Park, Kansas, USA) and secured to a heating pad (731-500-000R, Sunbeam, Boca Raton, Florida, USA). Mice were subcutaneously dorsally injected with 20 μL saline per gram of body weight (0.9% sodium chloride injection, USP, Hospira Inc, Lake Forest, Illinois, USA) to maintain hydration. Abdominal fur was removed with Nair hair removal body cream (NRSL-22339-07, Church and Dwight Co., Ewing, New Jersey, USA). Specifically, cotton tipped applicators (803-WC Hospital, Puritan Medical Products Company LLC, Guilford, Maine, USA) were used to first apply a generous amount of Nair to the fur. The Nair-covered fur was massaged lightly with the cotton tipped applicators for 5 min. The cotton tipped applicators were then used to remove the fur with wiping motion. Sterile alcohol prep pads (Cat. No.22-363-750, FisherBrand, Pittsburgh, Pennsylvania, USA) were then used to remove the remaining fur and Nair through wiping. The abdominal skin was then exfoliated with Nair for another 5 min with gentle massaging. After removing Nair with alcohol wipes, approximately 0.1 mL of the ampyrone solution (38% w / w, aqueous) was immediately applied with a cotton-tipped applicator before the residual alcohol on the skin had fully dried. The solution was then lightly massaged over an approximately 1 cm2area of skin for 5 min. It is estimated that only 20% or less of the ampyrone solution will make it into the skin, with the rest falling off or being absorbed into the cotton tipped applicator. The mouse belly was gently pressed with a glass slide (FisherBrand plain microscope slides, 75 x 50 x 1.0 mm, Pittsburgh, Pennsylvania, USA) to flatten features for imaging. Imaging was done with a Canon EOS Rebel T6 Digital SLR camera (DS126621, Canon, Tokyo, Japan) equipped with a Canon EFS 18-55 mm MACRO 0.25 m / 0.8 ft lens (Canon, Tokyo, Japan) illuminated by a portable photography light (MOLUS G60 60W portable photography light, Zhiyun, Guangxi, China) set to 6 W illumination power (10%) and 5500 K color temperature. Both the camera and illumination were linearly polarized and set at angle with respect to each other to eliminate reflection from the gel or glass slide.

[0216] After imaging, the mouse abdomen was rinsed with warm 5 mL PBS (PBS, 1X, Corning Inc., Corning, New York, USA) gently with a transfer pipette (Cat. No. 13-711-9AMMD, FisherBrand, Pittsburgh, Pennsylvania, USA) to extract the skin-absorbed ampyrone and reverse the transparency effect. Delicate task wipers (Kimberly-Clark, Irving, Texas, USA) were used to prevent the fur from getting wet to prevent hypothermia or infection. It is estimated that ~80% ofATTORNEY DOCKET NO.221910-2720 the skin-absorbed ampyrone was removed from the skin during this step. The mouse abdomen was then wiped with alcohol prep pads (Cat. No. 22-363-750, FisherBrand, Pittsburgh, Pennsylvania, USA) one more time before eye lubricant gel (Cat. No. B000URVDQ8, Genteal, Fort Worth, Texas, USA) was applied to hydrate the skin. A tough bioadhesive hydrogel (Sonopatch AD48, Sonologi, Palo Alto, California, USA) with a minimum 1 cm x 1 cm area was cut and placed onto the abdomen with the adhesive side touching the skin. The hydrogel pad was placed on the skin for 1 min with gentle pressure to ensure adhesion. The mouse abdomen was then wrapped with surgical tape (B014SPIK22, 3M Durapore 1” x 30’, Saint Paul, Minnesota, USA) to protect the hydrogel. It is essential that the tape be wrapped around itself such that the adhesive side of the tape touches itself. If the tape does not have two adhesive sides touching, it will come off when the mouse becomes active. The mouse was kept in a cage with bedding set on top of a heating pad (731-500-000R, Sunbeam, Boca Raton, Florida, USA), but with only half of the cage on the heating pad so that the mouse had the option to stay on the room temperature side of the cage.

[0217] For longitudinal abdominal skin imaging, the same recovery procedure as above was repeated for every day of imaging. The surgical tape and hydrogel were removed immediately before imaging, followed by reapplication of eye gel, hydrogel, and surgical tape. If imaging was not required every day, the hydrogel and surgical tape were only replaced after the mouse had damaged the surgical tape.

[0218] Achieving optical transparency for two photon excitation fluorescence imaging in the live mouse brain. Thy1-YFP-H mice (male and female, 3-4 weeks old, 9-15 g, C57BL / 6J [strain #000664] x B6.Cg-Tg(Thy1-YFP)HJrs / J [strain #003782]) from Jackson Labs were used for structural imaging of the brain. Mice were anesthetized with isoflurane with a custom 3D-printed anesthetic mask (SI Appendix, Dataset S1) and secured with a head holder (SGM-4, Narishige, Amityville, New York, USA). Mouse body temperature was maintained with a silicone rubber fiberglass flexible heater (SRFRA-8 / *, Omega Engineering, Norwalk, Connecticut, USA). Mice were subcutaneously injected dorsally with 20 μL saline per gram of body weight (0.9% sodium chloride injection, USP, Hospira Inc, Lake Forest, Illinois, USA) to maintain hydration. The scalp of the mouse was depilated and then exfoliated with Nair following the same procedure as above. Following exfoliation, ampyrone was applied to the scalp similarly to the abdomen until the scalp becomes visibly transparent to reveal the features in the skull such as the lambda, bregma, and other bone plate borders. A coverslip was secured to the top of the scalp above the visual cortex with a custom 3D-printed coverslip holder (SI Appendix, Dataset S2) to isolate the objective fromATTORNEY DOCKET NO.221910-2720 the remaining ampyrone solution, which acted as an index-matching medium between the coverslip and the transparent scalp. The coverslip holder provides pressure to the coverslip without obscuring the optical path of the microscope. This transparency window in the scalp can last up to 20 min before the effect gradually diminishes due to diffusion and clearance by systemic circulation. Reapplying ampyrone – by gently massaging the solution with a cotton applicator for 20 s either every 10–15 min or as needed – can effectively maintain the transparency in the scalp for an hour.

[0219] Two-photon excitation fluorescence imaging was done on a Prairie Ultima IV Two-photon In Vivo Microscope (Bruker, Billerica, Massachusetts, USA). A water immersion objective (20X Olympus XLUM Plan Fl W, 0.95 NA and 2.0 mm working distance, Olympus, Tokyo, Japan) was used.920 nm excitation (~110 mW incident power) was provided by a Mai Tai HP Deep See Ti- sapphire laser (Spectra Physics, Milpitas, California, USA) while emission was collected through a 525 / 50 nm bandpass filter. Pixel dwell time was 1.2 μs. Image size for the Z-stack was 512 x 512 pixels per frame with average number of 1 per frame and a z-step size of 5 μm. Field of view (FOV) for each image in the Z-stacks was 598 μm x 598 μm. Manual gain compensation was used for all Z-stacks.

[0220] For longitudinal YFP imaging, the same procedure was done as above except for modified imaging conditions and the recovery procedure (See Supporting Text for details). Specifically, mice were imaged while awake with the heating pad replaced with a treadmill to enable free mouse movement during imaging. To minimize motion artifacts and facilitate the consistent identification of the same blood vessel features across different days, a 10X air objective (10X Olympus UPlanFL N, 0.30 NA and 10 mm working distance, Olympus, Tokyo, Japan) was used. The stereotactic coordinates for the center of the FOV (in anterior-posterior (AP), medial-lateral (ML), dorsal-ventral (DV) coordinates with bregma set as zero) are (-3.2 mm, -2.3 mm, 0.2 mm). This corresponds to layer 1 of the primary visual cortex (V1). In addition to collecting the YFP emission with a 525 / 50 bandpass filter, the second harmonic generation (SHG) signal from collagen primarily in the scalp and skull was collected with a 460 / 50 bandpass filter to enable better z reference. The same 920 nm excitation with an incident power of ~110 mW was used for SHG in parallel with YFP. Pixel dwell time was 1.2 μs and image size for Z-stacks with a 5 μm step size was 512 x 512 pixels, with a FOV of 1.170 mm x 1.170 mm. Manual gain compensation was used for both the YFP and SHG channels independently.ATTORNEY DOCKET NO.221910-2720

[0221] Two mouse lines were used for calcium imaging. The first line was male and female Fostm2.1(icre / ERT2)Luo / J mice (Jax strain #: 030323) injected with 0.5-1 μL GCaMP8m AAV (AAV1-syn-jGCaMP8m-WPRE, titer: 2.4x1013vg / mL, Addgene # 162375-AAV1) into the center of the right hemisphere at P0 (0 days after birth). This line was used in FIGs. 10A-10D. The second line was male and female GCaMP6f mice (B6J.Cg-Gt(ROSA)26Sortm95.1(CAG- GCaMP6f)Hze / MwarJ [strain #028865] x B6.Cg-Tg(Camk2a-cre)T29-1Stl / J [strain #005359]). This line was used in FIG.10E. The Fostm2.1(icre / ERT2)Luo / J mice were 22 days old (P22) and the GCaMP6f mice were between 16 and 23 days old (P16 and P23). All mice were within the weight range of 8-15 g. The 20X objective and the same imaging parameters used for non- longitudinal YFP imaging were applied in FIGs.10A-10D in anesthetized mice. For FIG. 10E, different imaging parameters were used to capture the time-series data in awake mice. Specifically, to obtain the best firing activity and stimulus response, mice for time-series GCaMP imaging were imaged while awake, using the same awake imaging setup mentioned above for the longitudinal YFP imaging. The 10X objective described above was used to minimize motion artifacts and ensure at least some active and visible neurons were in the FOV. The excitation wavelength was 920 nm (~30 mW incident power) while emission was collected with a 525 / 50 bandpass filter. The image size was 256 x 256 pixels, dwell time was 12.4 μs, recording frame rate was 0.987 FPS, and FOV was 1.170 mm x 1.170 mm. The approximate stereotactic coordinates of the center of the FOV in FIG.10E (in AP, ML, DV coordinates with bregma set as zero) are (-1.2 mm, -0.9 mm, 0.3 mm). This location corresponds to layer 1 of the retrosplenial cortex (RSC). Vascular landmarks were checked with vascular maps of the mouse brain in literature to verify location. To provide stimulus, a hose was attached to the imaging setup and air was manually generated using a high pressure dust remover (Decon Labs Inc., Prussia, Pennsylvania, USA) to provide air pressure to both the mouse whiskers and face, promoting a running response on the treadmill. The RSC is responsible for integrating sensory and nonsensory information, with correlations between running speed and activity in the RSC reported. Full setups for both longitudinal and functional imaging are shown in FIGs.22A-22D. For both YFP and GCaMP imaging, the mice were less than 28 days old (P28) to ensure that the skull was not too thick for through-skull imaging. The reasoning behind this choice is that, although ampyrone effectively eliminates scattering caused by the scalp, it cannot penetrate or render the skull transparent. For 3D reconstruction from the z-stacks, IMARIS was used, with the 3D reconstructions processed by applying background correction, gamma correction, and a gaussian filter, followedATTORNEY DOCKET NO.221910-2720 by false coloring as well as the adjustment of brightness and contrast. To process the calcium data, images were aligned using the Enhanced Correlation Coefficient (ECC) algorithm from OpenCV. A translation motion model was used to align images with respect to the first image. A 50-element sliding median was used to calculate the baseline for baseline correction. To quantify time series changes in fluorescence intensity, ^^ was defined as the time-dependent baseline- corrected calcium signal and ^^^as the average signal from a low neural activity time window ofthe baseline-corrected signal when no stimulus was applied. Then ^^^^ was calculated as ^^^^ ൌ^^ െ ^^^.Example 6: Post-Treatment Characterization

[0222] Protein extraction from mouse skin. Age-matched C57BL / 6J mice (16 weeks old, 20-30 g; five males and females per group) were used for protein extraction experiments. Mice were massaged on the abdomen with the 38% w / w ampyrone aqueous solution for 5 min, while the control group received the same treatment with phosphate buffered saline (PBS, 1X, Corning Inc., Corning, New York, USA). The ampyrone or PBS solution then remained in contact with the skin for 20 min to match the approximate time window for imaging experiments. After treatment, all mice were subjected to transparency reversal and bioadhesive hydrogel application under the same conditions as abdominal imaging described above in “Achieving optical transparency for in vivo abdominal imaging.” Following a 72 h recovery period, all mice were humanely euthanized by cervical dislocation and the abdominal skin was excised from the mice. Prior to the digestion, the excised skin was washed twice with 1 mL of 1X PBS, followed by dermal-side-down soaking in Dispase II solution (Dispase II, Millpore Sigma D4693) at 5 mg / mL for 30-40 min at 37°C. The dermis was finely chopped and spread across a Petri dish, then subjected to enzymatic digestion in Liberase™ (Liberase™ Research Grade, Millipore Sigma #5401119001) at 1 mg / mL diluted in DMEM (DMEM, high glucose, pyruvate, Gibco, #11995073) for 20-30 min at 37°C with gentle agitation. DNase I (Deoxyribonuclease I from bovine pancreas, Millipore Sigma DN25) at 1 mg / mL was added to the mixture and incubated for 15 min with agitation ceasing after the DNase I was added. With DNase I incubation, the digested tissue was centrifuged at 4000 relative centrifugal force (RCF) for 5 min, supernatant was removed, and the pellet was washed twice with 1 mL of 1X PBS before resuspension in 500 mL of homogenizer buffer (T-PER Tissue Extraction Reagent, Thermo Scientific #78510) containing a 1:100 protease inhibitor cocktail (Protease Inhibitor Cocktail, Millipore Sigma P8340). Samples were frozen at -80°C for 12 h, then centrifuged at 5000 RCF for 10 min and the supernatant was transferred into low-bind protein tubes (Protein LoBind 1.5 mL Polypropylene Snap Cap Microcentrifuge Tubes, Eppendorf, #13-698-794). ProteinATTORNEY DOCKET NO.221910-2720 concentration was measured by bicinchoninic acid (BCA) protein assay (Pierce BCA Assay, Thermo Scientific #23225) and the samples were prepared for mass spectrometry.

[0223] Tandem mass spectrometry sample preparation. 20 μg resuspended epidermis protein extract for each of the samples were reduced in a solution of 5% v / v sodium dodecyl sulfate (15553027, Thermo Fisher Scientific, Waltham, Massachusetts, USA) with 10 mM dithiothreitol (Bio-Rad, 1610611, Hercules, California, USA) and incubated at 95°C for 10 min. Samples were then allowed to cool to room temperature and then alkylated using 40 mM 2-chloroacetamide (C0267-500G, Sigma-Aldrich, St. Louis, Missouri, USA) and incubated for 1 h at room temperature. The alkylation reaction was quenched by adding 85% m / v phosphoric acid (W290017-1KG-K, Millipore Sigma, Burlington, Massachusetts, USA) to a final concentration of 1.2% v / v phosphoric acid in the alkylated protein solution.700 μL (7X volume) of S-trap bind wash buffer, 100 mM triethylammonium bicarbonate buffer (TEAB, 60044974, Fisher Scientific, Hampton, New Hampshire, USA), and 90% v / v methanol (A452-4, Fisher Scientific, Hampton, New Hampshire, USA), was then added to the alkylated sample. Samples were then loaded onto a micro-S-trap-column (C02-micro-80, Protifi, Fairport, New York, USA) in 175 μL increments and spun at 4000 RCF for 20 s for each loading cycle. Sample caught on the column was then washed three times using the S-trap bind wash buffer with spins of 4000 RCF for 30 s to pass the buffer over the column. Mass spectrometry-grade trypsin (Promega V5113, Madison, Wisconsin, USA) was then diluted in 50 mM TEAB buffer (60044974, Fisher Scientific, Hampton, New Hampshire, USA) to a concentration of 0.04 mg / mL.25 μL of the diluted trypsin solution, or 1 μg of trypsin, was loaded onto each column to make a final trypsin:protein ratio of 1:20. Samples were then incubated at 47°C for 90 min. The column was then washed using 50 mM TEAB followed by 0.2% v / v formic acid (28905, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in water with a spin at 1000 RCF for 60 s between each wash. Digested samples were then eluted by adding 40 μL of a solution of 50% v / v acetonitrile (A955-212, Fisher Scientific, Hampton, New Hampshire, USA), 0.2% v / v formic acid, and 50% v / v water then spun at 4000 RCF for 60 s. Samples were then dried using vacuum centrifugation (CentriVap Complete Vacuum Concentrator, Labconco #7315023, Kansas City, Missouri, USA) for 16 h. The samples were resuspended in 0.2% (v / v) formic acid diluted in water immediately prior to mass spectrometry data acquisition.

[0224] Tandem mass spectrometry data acquisition. All samples were resuspended in a 0.2% v / v formic acid diluted in water at 1 μg / μl, and 1 μL (1 μg) of each sample was added to a new tube to make a pooled sample for the six gas phase fractionation injections. 1 μL (1 μg) of the total peptide solution was injected in the column for each sample. Peptides were separated over a 25ATTORNEY DOCKET NO.221910-2720 cm EasySpray reversed phase LC column (75 μm inner diameter packed with 2 μm, 100 Å, PepMap C18 particles, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The mobile phases (A: water with 0.2% v / v formic acid and B: acetonitrile with 0.2% v / v formic acid) were driven and controlled by a Dionex Ultimate 3000 RPLC nano system (ULTIM3000RSLCNANO, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Gradient elution was performed at 300 nL / min. Mobile phase B was increased from 1 to 5% v / v over 6 min, followed by a gradual increase to 25% v / v by 60 min, and ending with a ramp to 90% v / v B at 71 min, and a wash at 90% v / v B for 5 min. Flow was then ramped back to 1% v / v B over the course of 1 min, and the column was re-equilibrated at 1% v / v B for 15 min, for a total analysis of 90 min. Eluted peptides were analyzed on an Orbitrap Fusion Tribrid MS system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Precursors were ionized using an EASY-Spray ionization source (Thermo Fisher Scientific, Waltham, Massachusetts) source held at +2.2 kV compared to ground, and the column was held at 40^°C. The inlet capillary temperature was held at 275^°C. For gas phase fractionation injections, a pooled sample was injected six times and acquired using staggered-window injections with m / z windows of 395 to 505 m / z, 495 to 605 m / z, 595 to 705 m / z, 695 to 805 m / z, 795 to 905 m / z, and 895 to 1005 m / z. For all staggered-window injections a 4 m / z precursor isolation window was used. MS / MS scans were collected using high-energy collisional dissociation at 33 normalized collision energy and mass analysis was performed in the Orbitrap using a resolution of 30,000 while scanning from 120–2000 m / z. Individual samples were collected using single-injection data independent acquisitions (DIA) with survey scans of peptide precursors collected in the Orbitrap from 385-1015 m / z with an automatic gain control target of 400,000, a maximum injection time of 55 ms and a resolution of 60,000. An isolation window of 16 m / z was used to select precursor ions with the quadrupole. MS / MS scans were collected using high-energy collisional dissociation at 33 normalized collision energy and mass analysis was performed in the Orbitrap using a resolution of 30,000 while scanning from 120–2000 m / z.

[0225] Mass spectrometry database search and quantitation. Proteins were identified with DIA- NN and MSFragger 4.0 software on the Fragpipe GUI version 21.0, searching against the UniProt mouse reviewed proteome in addition to common contaminants. Methionine oxidation (+15.995 Da) and N-terminal acetylation (+42.011 Da) were included as variable modifications, while carbamidomethylation of cysteine (+57.021 Da) was included as a fixed modification. Precursor ion search tolerance was set to 20 ppm and fragment ion mass tolerance was set to 20 ppm. Peptide and protein identifications were thresholded at a 1% false discovery rate using a target- decoy method. Proteins were quantified and normalized by label-free quantitation using the DIA-ATTORNEY DOCKET NO.221910-2720 NN software. For quantitative comparisons, protein intensity values were log transformed, and missing values for proteins were imputed from a normal distribution with a width of 0.3 standard deviations and a down shift value of 1.8 standard deviations in Perseus version 2.0. A two-sided t-test was additionally done using Perseus with false discovery rate (FDR) correction performed using permutation-based FDR with 250 randomizations. Fold change was calculated in comparison with a PBS-treated control and log2 transformed for the volcano plot.

[0226] Animal subject preparation for tissue collection and histopathology. Age-matched C57BL / 6J (male and female, 16 weeks, 20-30 g) mice were used for all experiments.8 mice (4 male and 4 female mice) were depilated, exfoliated, treated with ampyrone in the abdominal skin, and subsequently reversed for the transparency effect as described in the “Achieving optical transparency for in vivo abdominal imaging” section. The mice were monitored and given the same recovery treatment with hydrogel as described above. The mice were then submitted to the Stanford Veterinary Service Center Necropsy Lab for histology, blood chemistry, and hematology assays after 24 h of recovery as detailed below in “Tissue collection and histopathology”. The same exact process was done on another 4 male and 4 female mice using phosphate buffered saline (PBS) as a control. The same process was repeated on another 16 mice (8 male and 8 female, evenly divided between the ampyrone group and the PBS control group with equal number of animals of each sex in each group), except these mice were submitted to the veterinary service after a 14-day recovery period.

[0227] Tissue collection and histopathology. Mice were euthanized by CO2asphyxiation and cardiac exsanguination. Terminal cardiac blood was collected for complete blood counts (CBC) and serum chemistry evaluation through the Stanford Animal Diagnostic Laboratory. The abdominal skin and subcutaneous tissues were collected and immersion fixed in 10% neutral buffered formalin for 72 h. Following fixation, two longitudinal sections of abdominal skin were collected along the ventral midline, parallel to hair follicle growth. Formalin-fixed skin samples were submitted to HistoTec Laboratories (Hayward, CA), processed routinely, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). H&E samples were evaluated by a board-certified veterinary pathologist using an ordinal scale (scores 0-4) where scores reflect the percentage of tissue exhibiting a given finding. Scores were assigned as follows: 0 = absent; 1= minimal (<25% of sample), 2 = mild (25-50% of sample), 3 = moderate (50-75% of sample), 4 = severe (>75% of sample). Skin samples were scored for the following parameters: mucosal ulceration; follicular and dermal necrosis + / - inflammation; superficial bacteria; acanthosis andATTORNEY DOCKET NO.221910-2720 hyperkeratosis; serocellular crusts; dermal fibrosis; follicular and adnexal dropout or disorganization; lymphoplasmacytic dermatitis.

[0228] Resolution characterization using phantoms containing fluorescent beads. For resolution characterization in FIGs. 19A-19D, imaging phantoms were prepared by adding 600 mg of ampyrone along with 10 mg of SeaPlaque agarose (50101, Lonza, Rockland, MO, USA) into 1 mL of water a 20 mL scintillation vial (DWK986546, Sigma-Aldrich, Burlington, MA, USA). The suspension was heated at 80°C for 10 min and then thoroughly mixed using a vortex mixer. It was then heated for another 10 min at 80°C and mixed again with a vortex mixer. A diluted suspension of fluorescent beads was prepared by diluting 1 μL of 200 nm diameter green fluorescent microspheres (FSDG002 / FS02F, 1% solids, Bangs Laboratories, Inc., Fishers, IN, USA) with 1 mL water. The PSF measured from these sub-diffraction fluorescent beads enables a continuous characterization of spatial frequencies, as the MTF can be obtained by taking the Fourier transform of the PSF.10 μL of this diluted suspension was then added to the mixture in the 20 mL scintillation vial above. Next, 300 μL of the suspension was cast into disposable base molds (7 mm × 7 mm × 5 mm, REF 22363552, Fisher Healthcare, Pittsburgh, PA, USA) and sealed with a coverslip to prevent evaporation. The samples were chilled on ice for 10 min to set the gel before proceeding. It is essential that ampyrone be added to the phantom to prevent diffusion of ampyrone out of the transparent scalp placed atop the phantom during imaging.

[0229] Ampyrone-treated transparent scalp and skull were prepared following the procedure outlined above in “Achieving optical transparency for two photon excitation fluorescence imaging in the live mouse brain” using Thy1-YFP-H mice (male and female, 3-4 weeks old, 9-15 g, C57BL / 6J [strain #000664] x B6.Cg-Tg(Thy1-YFP)HJrs / J [strain #003782]) from Jackson Labs. After achieving optical transparency of the scalp with ampyrone, the animals were humanely euthanized and 5 mm x 5 mm sections of scalp and skull were extracted. In order to maintain good contact between the scalp, skull, and the phantom, the scalp and skull were separately submerged in an aqueous mixture containing 600 mg of ampyrone, 10 mg of SeaPlaque agarose, and 1 mL water in a 20 mL scintillation vial. This mixture was allowed to partially set around the skull or scalp for 5 min on ice, yielding a semisolid agar. The skull-containing semisolid agar was first placed on top of a freshly prepared phantom containing fluorescent beads, with the semisolid nature of the agar ensuring direct contact between the skull and the underlying phantom. The scalp-containing semisolid agar was then placed on top of the skull-containing agar, ensuring proper alignment. Three samples were obtained from these steps: bare agar containing the fluorescent beads, skull on top of the agar containing fluorescent beads, and transparent scalpATTORNEY DOCKET NO.221910-2720 and skull on top of the agar containing fluorescent beads.

[0230] The same two-photon excited fluorescence imaging procedure was used as in “Achieving optical transparency for two photon excitation fluorescence imaging in the live mouse brain.” Slightly modified imaging parameters were used: The excitation wavelength was 920 nm while emission was collected with a 525 / 50 bandpass filter. The image size was 256 x 256 pixels, dwell time was 20.0 μs, and FOV was 50 μm x 50 μm. Image size for Z-stacks was 256 x 256 pixels per frame (corresponding to an FOV of 50 μm x 50 μm) with 2 averages per frame and a 5 μm step size.

[0231] Optimal age range of mice for through-scalp imaging. Precisely controlling the exact age of the mice is essential for achieving optimal scalp transparency. Based on this experience, the skull of mice younger than P15 severely aberrates the 920 nm excitation light required for two- photon microscopy, despite appearing visibly transparent. Since two-photon microscopy is more sensitive to excitation aberrations than emission aberrations, imaging through the skull of P14 and younger mice becomes challenging. After approximately P28, the skull becomes too thick, introducing severe aberrations that prevent cortical imaging. Because the present approach does not render the skull transparent, achieving optical transparency in the scalp alone is insufficient for microscopic imaging of the cortex through both the scalp and skull if the skull causes significant aberrations. Therefore, experiential guidance suggests that P14–P28 represents the optimal age range for through-scalp two-photon microscopy using the present scalp transparency approach.

[0232] Recovery from anesthesia and ampyrone. Ampyrone is an analgesic, so careful tuning of anesthetic dosage is crucial during animal experiments involving ampyrone. To minimize combined exposure, it is recommended to use inhalational (e.g., isoflurane) rather than intraperitoneal anesthesia (e.g., ketamine), as the former allows real-time dose adjustments during procedures. Isoflurane dosing and flow rate should be continuously monitored and adjusted based on visual assessment of the mouse’s heart and breathing rates.

[0233] For optimal workflow, it is suggested that one researcher should focus on skin treatment and imaging, while another manages anesthesia. Prolonged exposure to both ampyrone and anesthesia can hinder recovery. However, when ampyrone is administered without anesthesia for awake experiments, experience indicates that mice can tolerate over an hour of exposure without recovery issues.

[0234] That said, prolonged ampyrone application to the skin, even in awake mice, reduces the likelihood of full skin recovery. To optimize recovery, it is best to keep the skin transparency areaATTORNEY DOCKET NO.221910-2720 to approximately 1 cm2and duration to approximately 20-40 min.

[0235] Transparency effect in the mouse scalp. Unlike with the abdomen, reversing scalp transparency for longitudinal two-photon microscopy presented challenges, as it was not possible to apply a bioadhesive hydrogel patch and secure it with surgical tape. This difficulty arises from the difficulty in attaching any foreign object to a mouse’s head without the animal eventually removing it. Unlike chronic brain implants, which are directly cemented to the skull, the present approach preserves the scalp, making it an unstable platform for securing external materials.

[0236] As a result, Eucerin Advanced Repair Cream (NART #87955, Eucerin, Beiersdorf AG, Germany) was replied to the scalp every 30 minutes for the first 5 hours after ampyrone treatment, then every 8 hours thereafter. However, this treatment is less effective than the bioadhesive hydrogel, and the scalp remains partly translucent for several days following ampyrone treatment, reversal via thorough PBS rinsing, and Eucerin application.

[0237] Interpretation of histology results. After 1 day after treatment, mice in the control (PBS) and experimental (ampyrone) group exhibited similar levels of ulceration and dermal necrosis. At 14 days after treatment, mice in both groups exhibited similar levels of dermal fibrosis and follicular and adnexal drop out. It should be noted that while only the removal of the stratum corneum is necessary for the diffusion of ampyrone through the skin, these histological images show that some deeper layers of the epidermis are also partially disrupted. This inadvertent disruption results from excessive mechanical rubbing of the skin, which can be minimized or avoided by applying gentler force. Furthermore, the fact that skin treated with PBS shows similar or even greater levels of epidermal disruption than ampyrone suggests that ampyrone itself did not cause this damage.

[0238] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above- described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims. REFERENCES 1. Ahn, M et al, Electron push-pull effects on intramolecular charge transfer in perylene-based donor-acceptor compounds. J. Org. Chem.86, 403–413 (2021).ATTORNEY DOCKET NO.221910-2720 Arslan, H et al, Optical penetration depths and tluence distributions in chicken breast and liver tissues. Opt. Spectrosc.127, 763-768 (2019). Berezin, KV et al, Optical clearing of human skin: Molecular modeling and in vivo OCT study. J. Biophotonics 17, e202300354 (2024). Bohren, CF et al, Absorption and Scattering of Light by Small Particles.(John Wiley & Sons, 1998). Brodie, BB et al, The fate of aminopyrine (pyramidon) in man and methods for the estimation of aminopyrine and its metabolites in biological material. J. Pharmacol. Exp. Ther.99, 171– 184 (1950). Cai, R et al, Whole-mouse clearing and imaging at the cellular level with vDISCO. Nat. Protoc. 18, 1197-1242 (2023). Carneiro, I et al, A robust ex vivo method to evaluate the diffusion properties of agents in biological tissues. J. Biophotonics 12, e201800333 (2019). Carr, JA et al, Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared. Proc. Natl. Acad. Sci. U.S.A.115, 9080-9085 (2018). Cherkezyan, L et al, Targeted alteration of real and imaginary refractive index of biological cells by histological staining. Opt. Lett.37, 1601-1603 (2012). Cho, SW et al., Sounding out the dynamics: a concise review of high-speed photoacoustic microscopy. J. Biomed. Opt.29, S11521 (2024). Choi, B et al, Determination of chemical agent optical clearing potential using in vitro human skin. Lasers Swg. Med.36, 72-75 (2005). Chung, K et al, Structural and molecular interrogation of intact biological systems. Nature 491, 332-337 (2013). Costantini, I et al, In-vivo and ex-vivo optical clearing methods for biological tissues: Review. Biomed. Opt. Express 10, 5251-5267 (2019). Crocker, JC et al, Methods of digital video microscopy for colloidal studies. J. Colloid Interface Sci.179, 298-310 (1996). Damo, M et al., PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens. Nature 619, 151–159 (2023).ATTORNEY DOCKET NO.221910-2720 Demichev, V et al, DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods 17, 41–44 (2020). Dershowitz, LB et al, Anatomical and functional maturation of the mid-gestation human enteric nervous system. Nat. Commun.14, 2680 (2023). Dow Smith, F. Optical Image Evaluation and the Transfer Function. Appl. Opt., AO 2, 335– 350 (1963). Drury, R. Theory and Practice of Histotechnology. (1981). Elias, JE et al, Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007). Erttirk, A et al, Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat. Med.18, 166-171. Evangelidis, GD et al, Parametric image alignment using enhanced correlation coefficient maximization. IEEE Trans. Pattern Anal. Mach. Intell.30, 1858–1865 (2008). Fabian, J et al, Light absorption of organic colorants, 1980th Ed. (Springer, 1980). Feng, W et al, An optical clearing imaging window: Realization of mouse brain imaging and manipulation through scalp and skull. J. Cereb. Blood Flow Metab., 0271678X23 l 167729 (2023). Furness, J Structure of the Enteric Nervous System in The Enteric Nervous System, J. Furness, Ed. (John Wiley & Sons, 2007), pp.1-28. Ghanavati, S et al, Automatic anatomical labeling of the complete cerebral vasculature in mouse models. Neuroimage 95, 117–128 (2014). He, F. et al, Multimodal mapping of neural activity and cerebral blood flow reveals long-lasting neurovascular dissociations after small-scale strokes. Sci. Adv.6, eabal933 (2020). Heller, JM et al, Optical properties of liquid glycerol in the vacuum ultraviolet. Radial. Res.52, 25-31 (1972). Helmchen, F. et al, Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005). Hong, G et al, Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng.1, 0010 (2017).ATTORNEY DOCKET NO.221910-2720 Horstmeyer, R et al, Guidestar-assisted wavefront-shaping methods for focusing light into biological tissue. Nat. Photonics 9, 563-571 (2015). Horton, NG et al., In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat. Photonics 7, 205–209 (2013). Hu, Z et al., First-in-human liver-tumour surgery guided by multispectral fluorescence imaging in the visible and near-infrared-I / II windows. Nat Biomed Eng 4, 259–271 (2020). Iijima, K et al, Optical clearing of living brains with MAGICAL to extend i n vivo imaging. iScience 24, 101888 (2021). Jiang, S et al, Shedding light on neurons: optical approaches for neuromodulation. Natl Sci Rev 9, nwac007 (2022). Julien, O et al, Caspases and their substrates. Cell Death Differ.24, 1380–1389 (2017). Kong, AT et al, MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nat. Methods 14, 513–520 (2017). Konig, J. Chapter 2 - Food colour additives of synthetic origin in Colour Additives for Foods and Beverages, M. J. Scotter, Ed. (Woodhead Publishing, Oxford, 2015), pp.35- 60. Krzywinski, M et al, Power and sample size. Nat. Methods 10, 1139-1140 (2013). Li, D et al., Molecular engineering of NIR-II AIE luminogen excited at 1700 nm for ultradeep intravital brain two-photon fluorescence imaging. Adv. Funct. Mater.33, 2303967 (2023). Li, M et al, Refractive index modulation in monolayer molybdenum diselenide. Nano Lett.21, 7602-7608 (2021). Li, Y et al., High-speed two-photon microscopy with adaptive line-excitation. Optica 11, 1138– 1145 (2024). Lichtman, JW et al, Fluorescence microscopy. Nat. Methods 2, 910–919 (2005). Llewellyn, ME et al, Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans. Nature 454, 784- 788 (2008). Lobner, SA et al, Chlorophyll- and anthocyanin-rich cell organelles affect light scattering in apple skin. Photochem. Photobiol. Sci.21, 261-273 (2022). Lucarini, V et al, Kramers-Kronig Relations in Optical Materials Research. (Springer, 2005).ATTORNEY DOCKET NO.221910-2720 Malitson, IH. Interspecimen comparison of the refractive index of fused silica. J. Opt. Soc. Am. 55, 1205-1209 (1965). Matryba, P et al, Advances in ex situ tissue optical clearing. Laser Photonics Rev.13, 1800292 (2019). Mayer, EA et al, Gut / brain axis and the microbiota. J. Clin. Jnvestig.125, 926-938 (2015). Miller, DA et al., Enhanced penetration depth in optical coherence tomography and photoacoustic microscopy in vivo enabled by absorbing dye molecules. Optica 12, 24 (2025). Murakami, TC et al., A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat. Neurosci.21, 625–637 (2018). Nassau, K The physics and chemistry of color: The fifteen causes of color, 2nd Ed. (John Wiley & Sons, 2001). Ntziachristos, V Going deeper than microscopy: The optical imaging frontier in biology. Nat. Med.1, 603-014 (2010). Nudell, V et al, HYBRiD: Hydrogel-reinforced DISCO for clearing mammalian bodies. Nat. Methods 19, 479-485 (2022). Oliveira, LMC et al, The Optical Clearing Method: A New Toof for Clinical Practice and Biomedical Engineering. (Springer Nature, 2019). Ou, Z et al., Achieving optical transparency in live animals with absorbing molecules. Science 385, eadm6869 (2024). Pawley, J. Handbook of biological confocal microscopy, J. Pawley, Ed. (Springer, 2016). Peña-Blanco, A et al, Bax, Bak and beyond - mitochondrial performance in apoptosis. FEBS J.285, 416–431 (2018). Powell, A et al., Stable encoding of visual cues in the mouse retrosplenial cortex. Cereb. Cortex 30, 4424–4437 (2020). Price, HC et al, Sucrose diffusion in aqueous solution. Phys. Chem. Chem. Phys.18, 19207- 19216 (2016). Raffel, M et al, Particle Image Velocimetry: A Practical Guide. (Springer, 2018). Rakhilin, N et al, An intravital window to image the colon in real time. Nat. Commun.10, 5647 (2019).ATTORNEY DOCKET NO.221910-2720 Rassier, DE. Sarcomere mechanics in striated muscles: From molecules to sarcomeres to cells. Am. J. Physiol. Cell Physiol.313, CJ34--Gl45 (2017). Renier, N et al, Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165, 1789-1802 (2016). Ritsma, L et al, Surgical implantation of an abdominal imaging window for intravital microscopy. Nat. Protoc.8, 583-594 (2013). Rossi, J et al, Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis. Cell Metab.13, 195-204 (2011). Sai, T et al, Designing refractive index fluids using the Kramers-Kronig relations. Faraday Discuss.223, 136-144 (2020). Schmidt, EL et al., Near-infrared II fluorescence imaging. Nature Reviews Methods Primers 4, 23 (2024). Searle, BC et al., Generating high quality libraries for DIA MS with empirically corrected peptide predictions. Nat. Commun.11, 1548 (2020). Shim, H et al, Fundamental limits to the refractive index of transparent optical materials. Adv. Mater.33, 2103946 (2021). Song, G et al, A review of low-cost and portable optical coherence tomography. Prog. Biomed. Eng. (Bristol) 3, 032002 (2021). Spencer, NJ et al, Enteric nervous system: Sensory transduction, neural circuits and gastrointestinal motility. Nat. Rev. Gastroenterol. Hepatol.17, 338-351 (2020). Susaki, EA et al, Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726-739 (2014). Tainaka, K et al., Chemical landscape for tissue clearing based on hydrophilic reagents. Cell Rep.24, 2196–2210.e9 (2018). Talei Franzesi, G et al., In vivo optical clearing of mammalian brain. bioRxivorg 2024.09.05.611421 (2024). Tang, M et al, Far-field super-resolution chemical microscopy. Light Sci. Appl.12, 137 (2023). Tanzid, M et al, Absorption-induced image resolution enhancement in scattering media. ACS Photonics 3, 1787-1793 (2016).ATTORNEY DOCKET NO.221910-2720 Tanzid, M et al., Imaging through plasmonic nanoparticles. Proc. Natl. Acad. Sci. U. S. A.113, 5558–5563 (2016). Todorov, MI et al., Machine learning analysis of whole mouse brain vasculature. Nat. Methods 17, 442–449 (2020). Tsai, PS et al, Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J. Neurosci. 29, 14553-14570 (2009). Tuchin, V et al, Light propagation in tissues with controlled optical properties. J. Biomed. Opt. 2, 401-417 (1997). Tuchin, V Tissue optics and photonics: Light-tissue interaction II. J. Biomed. Photonics Eng. 2, 030201 (2016). Tuchin, VV Optical Clearing of Tissues and Blood. (SPIE Publications, 2005). Tuchin, VV Tissue optics and pbotonics: light-tissue interaction. J. Biomed. Photonics Eng.1, 98-134 (2015). Tyanova, S et al., The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016). Ueda, HR et al., Tissue clearing and its applications in neuroscience. Nat. Rev. Neurosci.21, 61–79 (2020). UniProt Consortium, UniProt: The universal protein knowledgebase in 2025. Nucleic Acids Res.53, D609–D617 (2025). Wang, T et al., Three-photon imaging of mouse brain structure and function through the intact skull. Nat. Methods 15, 789–792 (2018). Wen, X et al, In vivo skin optical clearing by glycerol solutions: mechanism. J. Biophotonics 3, 44–52 (2010). Westphal, D et al, Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ.21, 196–205 (2014). Whiteley, I et al, DIRECT, a low-cost system for high-speed, low-noise imaging of fluorescent bio-samples. Biomed. Opt. Express 14, 2565–2575 (2023).ATTORNEY DOCKET NO.221910-2720 Xiong, B et al., Precise cerebral vascular atlas in stereotaxic coordinates of whole mouse brain. Front. Neuroanat.11, 128 (2017). Yamashita, A et al., Indocyanine blue (ICB) as a functional alternative to indocyanine green (ICG) for enhanced 700 nm NIR imaging. Int. J. Mol. Sci.25, 13547 (2024). Yang, G et al, Transcranial two-photon imaging of synaptic structures in the cortex of awake head-restrained mice. Methods Mol. Biol.1010, 35–43 (2013). Yasir, M et al, Enhancing the refractive index of polymers with a plant-Based pigment. Small 11, 2103061 (2021). Yu, F. et al., Analysis of DIA proteomics data using MSFragger-DIA and FragPipe computational platform. Nat. Commun.14, 4154 (2023). Yun, SH et al, Light in diagnosis, therapy and surgery. Nat. Biomed. Eng.1, 0008 (2017). Zhao, YJ et al, Skull optical clearing window for in vivo imaging of the mouse cortex at synaptic resolution. Light Sci. Appl.7, 17153(2018).

Claims

ATTORNEY DOCKET NO.221907-2720 CLAIMS What is claimed is:

1. A method for imaging an organ or tissue in a subject, the method comprising: (a) administering a composition comprising a clearing compound to the subject, wherein an interaction between the clearing compound and at least one overlying tissue in the subject creates a transparent spectral window in the at least one overlying tissue; and (b) visualizing the organ or tissue through the at least one overlying tissue; wherein the transparent spectral window comprises the entire visible spectrum.

2. The method of claim 1, wherein the transparent spectral window is from about 380 nm to about 780 nm.

3. The method of claim 1, wherein the organ comprises a brain, a gastrointestinal tract, muscle, bones, blood vessels, a liver, a bladder, a spinal cord, a retina, a heart, a pancreas, a spleen, lungs, a trachea, kidneys, lymph nodes, a thymus, ovaries, testes, a uterus, a prostate or any combination thereof.

4. The method of claim 1, wherein the tissue comprises brain tissue, gastrointestinal tissue, muscle, connective tissue, bone tissue, blood vessels, liver tissue, bladder tissue, a tumor, spinal cord tissue, retinal tissue, heart tissue, pancreatic tissue, spleen tissue, lung tissue, tracheal tissue, kidney tissue, lymph node tissue, thymus tissue, ovary tissue, testes tissue, uterine tissue, prostate tissue or any combination thereof.

5. The method of claim 1, wherein the at least one overlying tissue comprises skin, muscle, epithelial tissue, connective tissue, or any combination thereof.

6. The method of claim 1, wherein the clearing is selected from ampyrone or sunset yellow.

7. The method of claim 1, wherein the subject is a mammal, a bird, a reptile, an amphibian, a fish, an arthropod, a mollusk, a cnidarian, an echinoderm, an annelid, a platyhelminthes, a nematode, or a plant.

8. The method of claim 7, wherein the mammal is a human, rat, mouse, rabbit, vole, tree shrew, guinea pig, hamster, cat, dog, pig, sheep, cow, or horse.

9. The method of claim 7, wherein the bird is a chicken, turkey, duck, parrot, or finch.

10. The method of claim 1, wherein the organ or tissue is visualized in situ in the subject.ATTORNEY DOCKET NO.221907-2720 11. The method of claim 1, wherein performing step (a) reduces light scattering between two or more tissue components, the tissue components having different refractive indices.

12. The method of claim 1, wherein performing step (a) increases light transmittance through the at least one overlying tissue by at least 50-fold compared to light transmittance through the at least one overlying tissue before performing the method.

13. The method of claim 1, wherein performing the method allows visualization of at least one feature in the subject at least about 200 μm below a skin surface of the subject.

14. The method of claim 1, wherein the composition comprises the clearing compound in a concentration of from about 30% (w / w) to about 45% (w / w) in an aqueous carrier.

15. The method of claim 14, wherein from about 50 μL to about 150 μL of the composition are administered per every 1 cm2of surface area of the overlying tissue.

16. The method of claim 1, wherein the composition is administered to the subject topically.

17. The method of claim 1, wherein the clearing compound is non-toxic.

18. The method of claim 1, wherein following visualizing, the clearing compound is excreted by the subject in less than about 10 hours.

19. The method of claim 1, further comprising removing at least a portion of the clearing compound from the subject following performing the method by wiping or rinsing the at least one overlying tissue.

20. The method of claim 1, wherein visualizing is accomplished using reflectance imaging, fluorescence imaging, laser speckle imaging, two-photon excitation spectroscopy, optical coherence tomography (OCT), light sheet microscopy, super-resolution microscopy, epifluorescence microscopy, fluorescence mediated tomography, photoacoustic tomography, three-photon microscopy, Brillouin microscopy, Raman microscopy, confocal microscopy, TIRF microscopy, brightfield / darkfield microscopy, DIC microscopy, structured illumination microscopy, or a combination thereof.

Images