Name: Transient Optical Clearing Using Absorbing Molecules for Ex Vivo and In Vivo Imaging (Video) | JoVE Journal
Uploaded: 2025-07-11T16:20:00.000+00:00
Duration: 7 min 15 s
Description: Discover transient optical clearing using tartrazine to enhance biomedical imaging in ex vivo tissues and live animals. This reversible method improves imaging depth and clarity for researchers.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

ZO acknowledges support from The University of Texas at Dallas.

All animal experiments conducted were approved by The University of Texas at Dallas&#39; Institutional Animal Care and Use Committee (IACUC) in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The reagents and the equipment used are listed in the Table of Materials. Image processing code is available at Github:&#160;https://github.com/ou-group-utd/JoVE-2025-Image-Processing
1. Tartrazine solution preparation
<ol>
	<li>For ex vivo samples
	<ol>
		<li>To prepare dye solutions, calculate the required mass of dye powder (Tartrazine) to prepare the desired volume of the solution. Use the formula: 
		Mass (g) = Molarity (M) &#215; Molecular Weight (g/mol) &#215; Volume (L) 
		NOTE: To calibrate actual molarity, measure the absorbance of prepared solutions using a UV-VIS spectrometer and apply the Beer-Lambert Law19. 
		&#8203;CAUTION: Use an analytical balance to weigh the precise amount of the powder. Wear appropriate PPE (personal protective equipment), including a lab coat, gloves, and safety goggles, when working with dye powders. Avoid inhalation or skin contact.</li>
		<li>Transfer the dye powder to a clean glass vial and add the required volume of deionized (DI) water using a volumetric pipette to ensure volumetric precision.</li>
		<li>If the powder does not fully dissolve at room temperature, place the glass vial in a laboratory oven or furnace preheated to 40 &#176;C and heat for 10 min or until powder is completely dissolved (Figure 1A). 
		NOTE: Low molarity solutions are completely soluble at room temperature and remain in liquid form, while high concentration (&#62;0.37 M) solutions require heating. 
		CAUTION: Excessive heating will evaporate water and alter the actual concentration of the solution.</li>
		<li>Stir on vortex mixer for 30 s to 1 min to ensure complete dissolution.</li>
		<li>To prepare a 0.65 M solution, dissolve 427.5 mg of Tartrazine powder in 1 mL of DI water19. For other concentrations, refer to Table 1. 
		NOTE: Dispose of unused dye solutions and contaminated materials according to standard chemical waste disposal procedures.</li>
	</ol>
	</li>
	<li>For in vivo samples
	<ol>
		<li>Calculate the required mass of low-melting temperature agarose microparticles and Tartrazine to prepare a 0.65 M concentration with 0.4% (4 mg/mL) agarose hydrogel solution. 
		NOTE: Follow standard safety precautions as described in step 1.1.1 when handling all reagents.</li>
		<li>Transfer the weighted powders into a clean glass vial. Add DI water using a volumetric pipette.</li>
		<li>Place the vial in a laboratory oven or furnace preheated to 75 &#176;C for approximately 10-15 min or until the agarose particles are completely dissolved.</li>
		<li>Occasionally mix using a vortex mixer during the heating process for uniform dissolution.</li>
		<li>Allow the solution to cool to room temperature before use. If it solidifies into gel form, use the vortex mixer for 30 s to re-liquefy it.</li>
		<li>To prepare a 0.65 M hydrogel solution, dissolve 427.5 mg of Tartrazine powder and 4 mg of agarose in 1 mL of DI water. To prepare solutions of other concentrations, maintain the agarose concentration at 0.4% (4&#8239;mg/mL) and refer to Table 1 for the corresponding Tartrazine mass17.</li>
	</ol>
	</li>
</ol>
2. Optical clearing of&#160;ex vivo tissues
<ol>
	<li>Sample preparation
	<ol>
		<li>To prepare ex vivo tissue, slice chicken breast tissue to the desired thickness parallel to the collagen fibers.</li>
		<li>Clean the sliced tissue gently with water.</li>
		<li>Measure thickness by placing the tissue between two clean microscope slides. Gently press the slides together and use a Vernier caliper to measure the distance between the slides at the center of the tissue.</li>
		<li>Soak the tissue in the dye solution. 
		CAUTION: Make sure that the volume of the solution is more than the sample volume and that the tissue is completely submerged.</li>
		<li>Place the container on an orbital shaker and shake at 100 revolutions per minute for 10 min.</li>
		<li>Let the tissue stay in the solution for 60 min at room temperature for low concentrations &#8804;0.4 M and at 40 &#176;C for higher concentrations &#62;0.4 M.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Remove the tissue from the dye solution using clean forceps.</li>
		<li>Place the sample between two transparent glass slides and capture the images using a digital or scientific camera with appropriate resolution and magnification settings.</li>
		<li>Conduct the imaging process in a controlled light environment to minimize external light interference.</li>
		<li>Repeat the imaging process for tissue samples with different thicknesses and dye solution concentrations (Figure 2B). 
		NOTE: Use an LED light source for back illumination. However, it works in the exact same way for a top illumination light source as well.</li>
	</ol>
	</li>
	<li>Optical transmission measurements
	<ol>
		<li>To measure the optical transmission, sandwich the chicken tissue between two transparent glass slides and measure the transmission using a UV-Visible-NIR Spectrometer (Figure 2C).</li>
		<li>Ensure that the system is baselined properly, and the light beam passes through the same region of the tissue before and after treating it with dye solutions to maintain consistency. 
		NOTE: Record the transmission spectra across a 400 nm to 2000 nm wavelength range, using a medium scan speed and a data interval of 1.0 nm. The full spectral scan takes approximately 5 min per sample.</li>
	</ol>
	</li>
</ol>
3. Optical transparency in live animals
<ol>
	<li>Animal preparation
	<ol>
		<li>Before the experiment, measure the animal&#39;s weight and note down the general activity level to establish a physiological baseline.</li>
		<li>Use a heating pad with the temperature set to 37 &#176;C to maintain the animal&#39;s body temperature during the experiment. 
		CAUTION: Continuously monitor the animal to prevent overheating.</li>
		<li>Administer anesthesia in accordance with institutionally approved protocols appropriate for abdominal and brain imaging.</li>
		<li>For inhalation anesthesia, induce with isoflurane at 1.5%-3% and flow rate 0.8 L/min for approximately 5 min before the imaging experiment and maintain it at 1%-2% throughout the experiment. 
		CAUTION: Monitor the animal&#39;s breathing pattern and depth continuously. Use a well-ventilated area to minimize anesthetic exposure to personnel.</li>
		<li>For injectable anesthesia, prepare a cocktail containing Ketamine HCL (16 mg/mL) and Dexmedetomidine HCL 0.2 mg/mL. Dose according to animal weight and approved institutional protocols.</li>
		<li>Trim the fur using small animal clippers from the desired imaging area.</li>
		<li>Apply depilatory cream to the trimmed skin area for less than 60 s to remove residual hair. Immediately remove the cream and clean the skin using alcohol wipes to avoid skin irritation.</li>
		<li>If injectable anesthesia with alpha-2 agonists (e.g., dexmedetomidine or xylazine) is used, administer atipamezole as the reversal agent following the experiment. Monitor the animal during recovery and ensure it returns to normal breathing and mobility before returning to its home cage.</li>
	</ol>
	</li>
	<li>Procedure for making tissue transparent
	<ol>
		<li>Prepare the imaging setup by placing a heating pad set to 37 &#176;C beneath the animal to prevent hypothermia. Align the light source and camera to capture the images.</li>
		<li>Use a 0.65 M&#160;Tartrazine hydrogel solution mixed with 0.3%-0.4% agarose gel for application (step 1.2).</li>
		<li>Apply the solution to the skin (belly or head, depending on the nature of the experiment) using a dropper and allow it to sit for a few seconds to initiate absorption (Figure 3A).</li>
		<li>Gently scrub the area using a cotton swab for even distribution of the dye solution and facilitate dermal absorption.</li>
		<li>Massage the area gently with a clean cotton swab and apply more solution till the skin starts showing transparency and internal organs are visible. During absorption, the skin will first show the appearance of orange-red dots, which gradually merge into a uniform color. This color change is a key visual indicator of adequate dye penetration and onset of optical clearing. 
		CAUTION: Ensure that the solution does not crystallize over the application area - crystallization can block skin passages, hindering the dye molecules&#39; absorption in the skin, which is highly crucial to achieve transparency. Sufficient absorption of dye molecules turns skin&#39;s color to orange-red.</li>
		<li>Continue massaging the area gently until the skin starts showing transparency and internal organs become visible. This process typically takes approximately 3-5 min of massaging for a small area, but duration may vary depending on animal size and skin thickness.</li>
		<li>Capture images using the aligned camera. Adjust focus, exposure, and white balance to optimize image quality.</li>
		<li>Gently place a glass slide or cover slip on the skin to minimize shine and reduce reflection for better quality images. 
		CAUTION: Handle all chemical solutions with proper personal protective equipment (lab coat, gloves, and eye protection), and dispose of used materials in accordance with the institution&#39;s chemical waste disposal protocols.</li>
	</ol>
	</li>
</ol>
4. Increasing organ visibility with image processing
<ol>
	<li>Image processing steps
	<ol>
		<li>Python with OpenCV is used for image processing. Import OpenCV, numpy, matplotlib, and PyWavelets.</li>
		<li>Load and Normalize the original Image. Load the original high-bit-depth .tif image. If any pixel value exceeds 255, normalize all pixels to the range [0, 255].</li>
		<li>Crop the Region of Interest (ROI). Invoke OpenCV&#39;s ROI selector to draw a bounding box around the organ area. Proceed only using the cropped version for all subsequent steps. 
		NOTE: Ensure that the bounding box tightly encloses the target area without cutting off any relevant structures.</li>
		<li>Split into LAB Channels. Convert the cropped color image to LAB color space and split into L(lightness), A, and B channels. 
		NOTE: Images are being processed in color, so the lab color space was used with image processing techniques being applied only to the L channel, where pixel intensities are located.</li>
		<li>Analyze Pixel Intensity Distribution. Plot a histogram of the L channel to assess contrast and intensity spread. 
		NOTE: Use the histogram to verify whether contrast enhancement is necessary and to guide parameter selection for contrast enhancement. You want your histogram to be spread across [0, 255].</li>
		<li>Apply CLAHE (Contrast Limited Adaptive Histogram Equalization) for local contrast enhancement. Create a CLAHE object with clipLimit = 5.0 and tileGridSize = (16,16). Execute CLAHE on the L channel and merge the CLAHE-enhanced L channel with the original A and B channels. Convert CLAHE from LAB to RGB for visualization if needed. 
		NOTE: If the histogram shows over-amplified noise (spikes), adjust &#39;clipLimit&#39; or &#39;tileGridSize&#39; and reapply to achieve balanced local contrast.</li>
		<li>Denoise the contrast-enhanced image. Perform a 2-level discrete wavelet decomposition on the normalized CLAHE L channel (scaled [0, 1]) with a soft threshold of 0.1. Reconstruct the denoised L channel, scale back to [0, 255], and merge the denoised L channel with the original A and B channels. Display the original cropped color image versus the wavelet-denoised result. 
		NOTE: Wavelet denoising reduces small fluctuations that might be misinterpreted as foreground in uniform regions.</li>
		<li>Segment organs of interests via Otsu&#39;s Adaptive Thresholding. Compute Otsu&#39;s threshold on the denoised L channel. Subtract a bias of 15 from the threshold to include low-intensity organ regions.
		<ol>
			<li>Apply a binary threshold with the bias value to obtain a mask of the organ pixels. Merge the mask with the original A and B channels and convert to RGB for viewing. 
			NOTE: The bias adjustment forces inclusion of organ regions beneath vessels. Re-tune bias if needed for a different dataset.</li>
		</ol>
		</li>
		<li>Proceed to analysis as desired.</li>
	</ol>
	</li>
</ol>

<ol><li>Yun, S. H., Kwok, S. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+in+diagnosis%2C+therapy+and+surgery%5BTitle%5D)+AND+%22Nat+Biomed+Eng%22%5BJournal%5D)">Light in diagnosis, therapy and surgery.</a> Nat Biomed Eng. 1 (1), 0008(2017).</li>
<li>Tuchin, V. V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTuchin%2C+VV+%22Optical+clearing+of+tissues+and+blood%22">Optical clearing of tissues and blood</a>. , SPIE Publications. (2005).</li>
<li>Ertürk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+3D+histology+powered+by+tissue+clearing%2C+omics+and+AI%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Deep 3D histology powered by tissue clearing, omics and AI.</a> Nat Methods. 21 (7), 1153-1165 (2024).</li>
<li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+molecular+interrogation+of+intact+biological+systems%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li>
<li>Costantini, I., Cicchi, R., Silvestri, L., Vanzi, F., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+and+ex+vivo+optical+clearing+methods+for+biological+tissues%3A+Review%5BTitle%5D)+AND+%22Biomed+Opt+Express%22%5BJournal%5D)">In vivo and ex vivo optical clearing methods for biological tissues: Review.</a> Biomed Opt Express. 10 (10), 5251-5267 (2019).</li>
<li>Genina, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+optical+clearing%3A+State+of+the+art+and+prospects%5BTitle%5D)+AND+%22Diagnostics%22%5BJournal%5D)">Tissue optical clearing: State of the art and prospects.</a> Diagnostics. 12 (7), 1534(2022).</li>
<li>Miller, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+penetration+depth+in+optical+coherence+tomography+and+photoacoustic+microscopy+in+vivo+enabled+by+absorbing+dye+molecules%5BTitle%5D)+AND+%22Optica%22%5BJournal%5D)">Enhanced penetration depth in optical coherence tomography and photoacoustic microscopy in vivo enabled by absorbing dye molecules.</a> Optica. 12 (1), 24-30 (2025).</li>
<li>Jia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tartrazine-enabled+optical+clearing+for+in+vivo+optical+resolution+photoacoustic+microscopy%5BTitle%5D)+AND+%22Biomed+Opt+Express%22%5BJournal%5D)">Tartrazine-enabled optical clearing for in vivo optical resolution photoacoustic microscopy.</a> Biomed Opt Express. 16 (6), 2504-2515 (2025).</li>
<li>Zuo, T., Tao, C., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absorbing+molecules+as+optical+clearing+agents+improve+the+resolution+and+sensitivity+of+photoacoustic+microscopy%5BTitle%5D)+AND+%22Opt+Lett%22%5BJournal%5D)">Absorbing molecules as optical clearing agents improve the resolution and sensitivity of photoacoustic microscopy.</a> Opt Lett. 50 (7), 2282-2285 (2025).</li>
<li>Narawane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+clearing+with+Tartrazine+enables+deep+transscleral+imaging+with+optical+coherence+tomography%5BTitle%5D)+AND+%22J+Biomed+Photonics+Eng%22%5BJournal%5D)">Optical clearing with Tartrazine enables deep transscleral imaging with optical coherence tomography.</a> J Biomed Photonics Eng. 29 (12), 120501(2024).</li>
<li>Ueda, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+clearing+and+its+applications+in+neuroscience%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Tissue clearing and its applications in neuroscience.</a> Nat Rev Neurosci. 21 (2), 61-79 (2020).</li>
<li>Mai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+cellular+mapping+in+mouse+using+standard+IgG+antibodies%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Whole-body cellular mapping in mouse using standard IgG antibodies.</a> Nat Biotechnol. 42, 617-627 (2024).</li>
<li>Ou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Achieving+optical+transparency+in+live+animals+with+absorbing+molecules%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Achieving optical transparency in live animals with absorbing molecules.</a> Science. 385 (6713), eadm6869(2024).</li>
<li>Sai, T., Saba, M., Dufresne, E. R., Steiner, U., Wilts, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designing+refractive+index+fluids+using+the+Kramers-Kronig+relations%5BTitle%5D)+AND+%22Faraday+Discuss%22%5BJournal%5D)">Designing refractive index fluids using the Kramers-Kronig relations.</a> Faraday Discuss. 223, 136-144 (2020).</li>
<li>Yu, T., Zhu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strongly+absorbing+molecules+make+tissue+transparent%3A+A+new+insight+for+understanding+tissue+optical+clearing%5BTitle%5D)+AND+%22Light+Sci+Appl%22%5BJournal%5D)">Strongly absorbing molecules make tissue transparent: A new insight for understanding tissue optical clearing.</a> Light Sci Appl. 14 (1), 10(2025).</li>
<li>Drexler, W., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDrexler%2C+W+%22Optical+coherence+tomography%3A+Technology+and+applications%22">Optical coherence tomography: Technology and applications</a>. Drexler, W., Fujimoto, J. G. , Springer International Publishing. Cham. 277-318 (2015).</li>
<li>Wang, F., Zhou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transparent+mice%27%3A+Deep-tissue+live+imaging+using+food+dyes%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">Transparent mice': Deep-tissue live imaging using food dyes.</a> Commun Biol. 7 (1), 1307(2024).</li>
<li>Yu, H., Liu, J. -B., Ma, Y. -S., Fu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transparent+live+mice+offer+more+possibilities+for+biomedical+research%5BTitle%5D)+AND+%22Innov+Med%22%5BJournal%5D)">Transparent live mice offer more possibilities for biomedical research.</a> Innov Med. 2 (4), 100098(2024).</li>
<li>Shabbir, M. W., Biswas, S., Kajla, R., Nadella, S., Ou, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designing+refractive+index+fluids+of+food+dye+for+light+propagation+through+scattering+media%5BTitle%5D)+AND+%22MRS+Commun%22%5BJournal%5D)">Designing refractive index fluids of food dye for light propagation through scattering media.</a> MRS Commun. , (2025).</li>
<li>Scheele, C. L. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiphoton+intravital+microscopy+of+rodents%5BTitle%5D)+AND+%22Nat+Rev+Methods+Primers%22%5BJournal%5D)">Multiphoton intravital microscopy of rodents.</a> Nat Rev Methods Primers. 2 (1), 89(2022).</li>
<li>Schmidt, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Near-infrared+II+fluorescence+imaging%5BTitle%5D)+AND+%22Nat+Rev+Methods+Primers%22%5BJournal%5D)">Near-infrared II fluorescence imaging.</a> Nat Rev Methods Primers. 4 (1), 23(2024).</li>
</ol>

All authors declare no conflict of interest.

This work presents a comprehensive and reproducible protocol method to achieve optical transparency in biological tissues utilizing absorbing molecules, specifically FDA-approved food dye Tartrazine (approved for use in food products, not in medical applications). This method effectively minimizes intrinsic scattering in tissue by altering the refractive index within the tissue's microenvironment, which enhances optical transparency. This study demonstrated the effectiveness of this method through both ex vivo a...

Biological imaging has long been challenged by the scattering of light, which obscures structures of interest1. Optical clearing techniques aim to reduce light scattering and enhance visual clarity, enabling the visualization of previously hidden structures2. The development of these techniques has led to a diverse array of methods designed to improve optical imaging of ex vivo biological tissues, even whole animals, without physical dissection of the samples3. However, the development of similar techniques for in vivo applications has been challenging. Techniques such as CLARITY Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / in situ-hybridization-compatible Tissue hYdrogel), offer high-resolution imaging but lack reversibility, making them unsuitable for live animal studies4. Constantini et al. have shown slower progress in&#160;in vivo imaging compared to ex vivo imaging clarity, which is due to the complexity of maintaining tissue viability while avoiding toxic levels of agents needed for tissue transparency5. Genina emphasizes the future of optical tissue clearing as reliant on non-invasive and reversible methods6. Recent studies have demonstrated the effectiveness of water-soluble absorbing dyes for in vivo optical clearing. For example, Miller et al. showed that topically applying Tartrazine significantly enhanced imaging depth in both OCT and photoacoustic microscopy (PAM) of live mouse skin, by reducing scattering and increasing the refractive index of interstitial tissue components7. Jia et al. used a 15% w/w Tartrazine solution to enhance in vivo PAM imaging of mouse ear and abdominal tissue, revealing fine microvascular structures with subcellular resolution that were otherwise obscured8. Zuo et al. further demonstrated brain imaging through intact scalp and skull in live mice using 0.6 M Tartrazine, achieving four times higher signal intensity and 3.5 times improved resolution compared to untreated tissue9. In another application, Narawane et al. enabled deep transscleral OCT imaging in porcine eyes by applying Tartrazine, achieving clear visualization of the suprachoroidal space, which was previously inaccessible due to strong scleral scattering10. Although ex vivo tissue clearing methods like CLARITY and iDISCO (Immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) achieve high optical transparency and subcellular resolution through lipid removal or solvent-based clearing, they are often unsuitable for in vivo imaging with live animals4,11,12. This limitation affects dynamic imaging, which is crucial for biomedical imaging of dynamic processes inside living animals, such as disease development and therapeutics.
Absorbing molecules with high aqueous solubility and distinct absorption band shapes, such as Tartrazine, a food-grade dye, offer promising advantages for achieving optical transparency in living animals13. By specifically introducing Tartrazine, the tissue selectively absorbs ultraviolet and blue wavelengths due to the dye&#39;s unique spectral characteristics, resulting in a modified refractive index profile that reduces scattering at longer wavelengths. This counterintuitive optical tissue clearing technique is optimized for in vivo imaging, and the Kramers-Kronig relations provide a scientific foundation for the observed transparency when using strongly absorbing molecules like Tartrazine14,15. For in vivo imaging, Tartrazine is combined with heated agarose particles and water to create a cohesive solution that adheres to skin better. The water-soluble nature of Tartrazine also facilitates easy removal of the dye-based solution from live animals, reducing potential effects of extended exposure to dye solutions. Additionally, the method has been adapted for various biomedical applications to improve the imaging depth, resolution, as well as sensitivity, including optical coherence tomography7,16 and photoacoustic imaging7,9, demonstrating its potential for the broader biomedical imaging community.
With the growing interest in the application of absorbing molecules in biomedical imaging17,18, the protocol presents a detailed procedure of utilizing food dye for ex vivo and in vivo applications, along with image processing and reversible in vivo imaging. This protocol is applied to chicken breast tissue to demonstrate ex vivo transparency and is then modified for in vivo head and abdominal imaging of mice, showing reversibility. A solution applied to mouse skin in vivo creates temporary, reversible transparency without harming the skin or tissue integrity. This technique can be integrated with an image processing pipeline, including denoising, to create an optimized imaging protocol for in vivo optical imaging of internal organs. When combined, these techniques enable better non-invasive, longitudinal imaging for real-time observations of biological processes. The use of safer agents that demonstrate reversibility overcomes previous limitations and provides a more suitable method for dynamic biological imaging.

<table><tbody><tr><td>Analytical Balance</td><td>HZK</td><td>FA220S</td><td></td></tr><tr><td>Animal anesthesia Machine</td><td>RWD life science co</td><td>TAIJI-IE</td><td>Inhalation Anesthetics</td></tr><tr><td>Atipamezole HCL injection 5 mg/mL</td><td>Covertus</td><td>82124</td><td>Injectable anesthesia</td></tr><tr><td>CMOS camera</td><td>Thor labs</td><td>CS165CU/M, Zelux</td><td></td></tr><tr><td>Dexmedetomidine HCL injection 5 mg/10 mL</td><td>Covertus</td><td>72960</td><td>Injectable anesthesia</td></tr><tr><td>Fiber Light Illuminator</td><td>RWD life science co</td><td>76301</td><td>Light source for top illumination</td></tr><tr><td>Glass slides</td><td>CravitySci</td><td>0303-2122</td><td>Super grade microscope slides</td></tr><tr><td>Homeothermic Blanket</td><td>RWD life science co</td><td>69027</td><td>Animal temperature controller</td></tr><tr><td>Image Processing</td><td>OpenCV</td><td>Python</td><td>Software</td></tr><tr><td>Insulin Syringe 0.5 mL 29 G</td><td>Comfort point</td><td>26028</td><td>Injectable anesthesia</td></tr><tr><td>Isoflurane</td><td>Covertus</td><td>SKU 029405</td><td>Inhalation Anesthetics</td></tr><tr><td>Ketmine HCL injection 100 mg/mL</td><td>Dechra</td><td>20042240</td><td>Injectable anesthesia</td></tr><tr><td>LED light source</td><td>GGE corp</td><td>TLB-01</td><td>Light source for top illumination</td></tr><tr><td>Lens</td><td>Thor labs</td><td>MVL4WA</td><td>3.5 mm focal length and f/1.4 aperture</td></tr><tr><td>Nair</td><td>Church &amp;amp; Dwight</td><td>L 1224</td><td>Hair removal Cream</td></tr><tr><td>PBS ph 7.4 (1x)</td><td>Gibco</td><td>2902869</td><td>Injectable anesthesia</td></tr><tr><td>Q-Tip</td><td>Puritan medical products company</td><td>806-wc hospital</td><td></td></tr><tr><td>SeaPlaque Agarose</td><td>Lonza</td><td>50101</td><td>Chemical</td></tr><tr><td>Tartrazine</td><td>MilliporeSigma</td><td>CAS number: 1934-21-0</td><td>Chemical</td></tr><tr><td>Transfer pipette</td><td>Fisherbrand</td><td>13-711-9AMMD</td><td></td></tr><tr><td>UV-VIS-NIR Spectrophotometer</td><td>Shimadzu</td><td>SolidSpec-3700i</td><td>Instrument</td></tr><tr><td>Vials</td><td>Qorpak</td><td>242633</td><td></td></tr><tr><td>Volumetric pipettes</td><td>Eppendorf research plus</td><td>3123000063</td><td>100-1000&amp;micro;L</td></tr><tr><td>Water Purification System</td><td>Avidity science</td><td>SOLO S</td><td>Instrument</td></tr></tbody></table>

transient optical clearing using absorbing molecules for ex vivo and in vivo imaging

Transient Optical Clearing Using Absorbing Molecules for Ex Vivo and In Vivo Imaging

Dynamic imaging is a foundational tool in biology and medicine, but it is limited by light scattering in live tissues caused by refractive index mismatches, which reduces penetration depth and resolution in biological tissues. Previous work introduced a novel approach to achieve optical transparency in live animals by utilizing strongly absorbing molecules. When these absorbing molecules, such as Tartrazine, an FDA-approved dye commonly used in foods, dissolve in water, they modify the refractive index of the aqueous medium through Kramers-Kronig relations. This modulation allows reduction of the refractive index mismatch between separate components in tissue with distinct refractive indices, thus mitigating scattering. This article reports the detailed methodology of the tissue clearing technique using a Tartrazine-based aqueous solution, applied to both ex vivo and in vivo samples. This approach reversibly renders live mouse bodies transparent in the visible spectrum, enabling clearer and deeper imaging of internal processes and structures. An advanced image processing algorithm was then used to improve the contrast and segmentation of organs of interest. Additionally, high transparency in ex vivo samples, such as chicken breast,&#160;treated with Tartrazine-based solution, is shown. This method offers a versatile and accessible approach to enhance&#160;imaging capabilities across various modalities and paves the way for advancement in the understanding of complex biological systems.

Demonstration of optical transparency in ex vivo chicken breast tissue 
To investigate light propagation through ex vivo tissue, we used sliced pieces of raw boneless chicken breast with thicknesses of 1.5 mm, 2.5 mm, and 3.5 mm (Figure 2B). The tissues were cut parallel to collagen fibers, ensuring consistent muscle fiber orientation. Images were taken using both a mesh grid pattern and ...

Optical clearing techniques, such as absorbing molecules solutions, reduce light scattering temporarily to enhance biological imaging and offer biocompatible, reversible in vivo imaging strategies for deeply embedded organs. This protocol presents detailed experimental procedures as well as advanced image processing to achieve high-quality, dynamic imaging for real-time dynamics in living animals.

Dynamic imaging is a foundational tool in biology and medicine, but it is limited by light scattering in live tissues caused by refractive index ...

This research aims to evaluate and implement tissue clearing using absorbing molecules as a non-invasive strategy to enhance biomedical imaging in both ex vivo tissues and live animals. We have demonstrated that the food dye tartrazine can be used to achieve rapid and reversible optical clearing in both tissues and live animals, significantly enhancing imaging depth and clarity. Our protocol addresses a critical gap in the reproducibility of transient optical clearing by observing molecules, and incorporates advanced computational visualization techniques to enhance feature visibility.By providing a detailed protocol for tissue clearing with absorbing molecules, our work helps the broader biology and biomedical imaging research community to apply these methods in their own studies. Our lab has been advancing this technology by exploring the physical interaction between light and tissue, expanding the range of available materials and developing optimized optical imaging platforms. To begin, calculate the required mass of tartrazine dye powder to prepare the desired solution volume.Transfer the calculated amount of dye powder to a clean glass vial. Using a volumetric pipette, add the required volume of deionized water to the vial to maintain precision. If the dye does not fully dissolve at room temperature, place the glass vial in a laboratory oven preheated to 40 degrees Celsius for 10 minutes or until completely dissolved.Then vortex the dye solution for 30 seconds to one minute to ensure complete dissolution. For the ex vivo tissue, slice a chicken breast tissue to the desired thickness in a direction parallel to the collagen fibers, and clean the sliced tissue gently with water. Place the tissue between two clean microscope slides to measure the thickness.Gently press the slides together and use a vernier caliber to measure the distance at the center of the tissue. Next, soak the tissue in the prepared dye solution. And place the container on an orbital shaker and shake at 100 revolutions per minute for 10 minutes.Remove the tissue from the dye solution using clean forceps. Place the sample between two transparent glass slides and capture images using a digital or scientific camera set to the appropriate resolution and magnification. Conduct the imaging process in a controlled light environment to minimize external light interference.To measure optical transmission, place the prepared sample in a UV-visible near-infrared spectrometer. Ensure the system is properly baselined and that the light beam passes through the same tissue region before and after treatment with dye solution to maintain measurement consistency. For the procedure, position the anesthetized animal on a heating pad tempered at 37 degrees Celsius.Using small animal clippers, trim the fur from the desired imaging area. Apply depilatory cream to the trimmed area for less than 60 seconds to remove any residual hair and immediately remove the cream. Clean the skin using alcohol wipes to prevent irritation.To prepare the imaging setup, place a heating pad set to 37 degrees Celsius beneath the animal to prevent hypothermia. Align the light source and camera to capture high-quality images. Use a 0.65 molar tartrazine solution mixed with 0.3%to 0.4%agarose gel for application.Using a dropper, apply the prepared solution to the skin, either on the belly or head, depending on the experiment. Allow the solution to sit for a few seconds to begin absorption. Now, with a cotton swab, gently scrub the application area to evenly distribute the solution and enhance dermal absorption.Then massage the area gently with a clean cotton swab and apply more solution as required. Observe for the appearance of orange red dots, which will gradually merge into a uniform color, indicating successful dye penetration and the start of optical clearing. Continue massaging the area until the skin becomes transparent and internal organs are visible, which usually takes around three to five minutes for a small area.Gently place a glass slide or cover slip over the skin to reduce surface shine and minimize reflection. Gently clean the dye solution from the skin using a cotton swab or soft cloth moistened with water. The optical clearing effect will reverse and the skin will gradually return to its original opaque appearance.After turning off the gas anesthesia, allow the animal to regain consciousness. Once the animal is fully awake and mobile, return it to its home cage. In untreated ex vivo chicken breast tissue, the underlying patterns were not visible, and spectrometer data showed low transmission values that decreased with increasing tissue thickness.Following treatment with a 0.65-molar tartrazine solution, the tissues became visibly transparent and the underlying grid and logo patterns emerged clearly. Substantial increases in transmission values across all thicknesses in the visible spectrum were also observed. In live mice, application of 0.65 molar tartrazine solution enabled visualization of internal organs through the skin within minutes of treatment, showing visibly increased transparency in abdominal regions.Optical clearing of the scalp allowed visualization of cerebral blood vessels without surgical removal of skin, demonstrating the technique's effectiveness for brain imaging. The clearing effect was fully reversible. Rinsing the tissue with water restored opacity and normal skin appearance returned within hours, with hair regrowth observed over several days.

transient-optical-clearing-using-absorbing-molecules-for-ex-vivo-vivo

Research

JoVE Journal

Bioengineering

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

Optical Clearing of Plant Tissues for Fluorescence Imaging

This protocol combines optical clearing and immunolabeling for full-volume confocal imaging of whole-mount retinal organoids. It preserves 3D structure, enabling detailed visualization of key neuronal pathways, improving the study of retinal maturation, spatial organization, and its potential applications in disease modeling and personalized medicine.

Whole-mount Retinal Organoid Visualization with Cellular Resolution

Discover transient optical clearing using tartrazine to enhance biomedical imaging in ex vivo tissues and live animals. This reversible method improves imaging depth and clarity for researchers.

Transient Optical Clearing Using Absorbing Molecules for Ex Vivo and In Vivo Imaging

Explore More Videos