A reversible approach to achieving optical transparency in live animals

This is a detailed protocol to help any interested person or team in achieving optical transparency in live animals in a reversible and minimally invasive manner:

1. Preparing an aqueous solution or gel of tartrazine:

1.1. Preparation of tartrazine solutions. A 30 w/w% tartrazine solution (equivalent to 0.62 M, accounting for volume expansion of the solvent, water) is used here as an example. Weigh 3.0 g of tartrazine and transfer it into a 20-mL scintillation vial. Add 7.0 mL of distilled water to the vial and vortex the mixture thoroughly. Place the vial in an oven preheated to 70-80°C and allow it to sit for 10 minutes. After this time, remove the vial from the oven; the tartrazine should be completely dissolved. Allow the solution to cool naturally to room temperature. The 30 w/w% tartrazine solution is stable at room temperature for 4 hours. It will gradually precipitate after 4 hours, but the mixture can be reheated in the 70-80°C oven to yield a homogeneous solution again. To prevent precipitation, this solution should be stored in a temperature-controlled oven, beam warmer, or orbital shaker set to 37°C.

1.2. Preparation of tartrazine gels. A 30 w/w% tartrazine gel (equivalent to 0.62 M, accounting for volume expansion of the solvent, water) is used here as an example. Begin with the 30 w/w% tartrazine solution from section 1.1 after it has been immediately removed from the 70-80°C oven, ensuring that all solids have completely dissolved to yield a homogeneous solution. Weigh 60 mg of agarose and add it to the tartrazine solution, ensuring no agarose powder adheres to the walls of the vial so that all of the agarose is immersed in the solution. DO NOT vortex the solution. Place the vial back into the 70-80°C oven and allow it to sit for 10 minutes. After this time, remove the vial from the oven; the agarose should be completely dissolved. Gently shake the vial to ensure the agarose is uniformly distributed in the solution. At this point, the mixture should remain a homogeneous solution. Place the vial in a 4°C refrigerator and allow it to sit for 10 minutes. After this time, remove the vial from the refrigerator; the solution should have turned into a clear, red gel. The 30 w/w% tartrazine gel is stable at room temperature for at least 24 hours.

1.3. Preparation of solutions and gels composed of other absorbing molecules. A similar procedure can be followed to prepare the solutions and gels composed of other absorbing molecules described in the “Topical application of dye solutions on live mice” section of the paper, which can yield less colored transparency to enhance the visibility

2. Achieve ex vivo tissue transparency with solutions of absorbing molecules:

2.1. Preparation of ex vivo tissue samples. Dissect ex vivo tissue samples (e.g., muscle, skin, intestines) from experimental animals in accordance with your APLAC protocol, and slice them into 1–2 mm sections if they are thicker than 2 mm. Paraformaldehyde fixation is not required but will not interfere with achieving the transparency effect. Place the sample flat at the bottom of a transparent tray or petri dish. Position the transparent container with the tissue sample over a printout of a grid pattern or other design, and place this setup on top of a small portable light table.

2.2. Observing ex vivo tissue transparency. Transfer the transparent container with the tissue sample into a temperature-controlled orbital shaker (such as this one). Set the temperature to 37°C without using the shaking function. Slowly pour the solution (not the gel!) prepared from sections 1.1 or 1.3 above, containing the absorbing molecules, into the tray or dish to completely submerge the tissue in the liquid. If the tissue starts to float, ensure that it is taped or pinned down to the bottom of the container to ensure full exposure to the solution. Depending on the thickness of the tissue, transparency should start to become visible within 15 minutes to 1 hour of immersion.

2.3. Observing transparency in other ex vivo tissue samples. Although we have not tested these samples in our lab, the U.S. National Science Foundation has demonstrated that freshly cut, thicker chicken muscle (>3 mm) can become more transparent by applying a tartrazine solution to the surface. You can read their report here. Additionally, the Manton Lab at the University of Cambridge achieved transparency in a neural organoid soaked in a 0.5 M tartrazine solution. Their results can be found here.

3. Achieve in vivo tissue transparency with gels of absorbing molecules:

3.1. Preparation of animal subjects. For in vivo experiments on mouse skin, 3- to 4-week-old mice on a C57Bl/6 background, weighing 10~15 g, should be used if your goal is to achieve transparent skin that reveals underlying internal organs visible to the naked eye. Any deviation from these specifics may result in insufficient visibility of the abdominal organs due to the amount of fat tissue beneath the skin and the varying diffusion properties of the skin. Please note that these are the animals used in the paper to visualize abdominal organs (“young (10 to 15 g) female thy1-YFP-H and C57BL/6J mice (for brightfield imaging of abdominal organs, 3 to 4 weeks old, Jackson Laboratory)”, see the “Vertebrate animal subject” section of the paper). 

Use inhalational anesthesia or intraperitoneal injection in accordance with your APLAC protocol to anesthetize the animal. Begin by generously applying Nair to the abdominal region of interest. After 5 minutes, remove the hair and Nair using alcohol pads. Reapply Nair, wait another 5 minutes, and remove it again with alcohol pads. Make sure not to leave Nair or any depilatory cream on the skin for more than 10 minutes (instructions, reference). The depilated skin should appear pink to achieve the best effect. Some animals within this age range may exhibit birthmarks or develop darker skin pigmentation with visible hair follicles embedded in the skin that cannot be removed by Nair. It is recommended to avoid using these animals.

3.2. Applying the gel on the skin. Use a Q-tip to apply the gel composed of absorbing molecules, as described in sections 1.1 or 1.3 above. Dip the Q-tip into the gel and transfer some onto the depilated skin, rubbing it repeatedly on the skin. You may also use your other hand to stretch the abdominal skin with your fingers to aid penetration. Ensure the gel does not crystallize over the application area (though the edges may show some yellow to orange powder from the dye molecule due to drying). Crystallization can block the skin’s passages and prevent molecule penetration, so check the gel’s stability by referring to section 1.1. After massaging the skin with the gel for 5 minutes, the treated area should appear orange-to-red with increased translucency. Continue massaging for an additional 5 minutes to achieve maximum transparency. Adequate absorption of the dye molecule is essential for transparency, so check the skin absorption by wiping off the gel. If the skin remains pink (its natural color) or turns yellow (rather than orange-to-red), this indicates insufficient absorption of the dye. In such cases, continue massaging or refer to the alternative method outlined in section 3.3 for improved results.

3.3. Enhancing the penetration of dye molecules. For mice with thicker and less permeable skin (commonly observed in C57Bl/6 mice weighing more than 15 g and in nude mice of all ages, thus deviating from our recommended mice in 3.1.), physical percutaneous enhancers may be helpful. One option is to mix cosmetic grade pumice with the gel made at approximately a 1:3 volume ratio before applying it to the skin with a Q-tip. Dip the Q-tip into the gel-pumice mixture and transfer some onto the depilated skin, rubbing it repeatedly on the skin. Ensure the dye molecules do not crystallize from the gel over the application area. After massaging the skin with the gel-pumice mixture for 5 minutes, the treated area should appear orange-to-red with increased translucency. Continue massaging for an additional 5 minutes to achieve maximum transparency. Adequate absorption of the dye molecule is essential for transparency, so always check the skin absorption by wiping off the gel. 

Another approach is to use a topical microneedle applicator to deliver the solution prepared in sections 1.2 or 1.3. Roll the microneedle applicator over the skin repeatedly, applying gentle pressure to facilitate the transdermal delivery of the absorbing molecules. After rolling the solution onto the skin for 5 minutes, the treated area should take on an orange-to-red hue with increased translucency. Continue rolling for an additional 5 minutes to maximize transparency. You may pause during the rolling process to gently massage the skin with your fingers.

3.4. Troubleshooting abdominal transparency. Sometimes, the absence of obvious features in the abdomen after the skin turns red is not due to a lack of skin transparency, but rather the presence of fat tissue beneath the skin (usually observed in mice weighing >15 g), which this approach cannot render transparent. To determine whether the issue lies with the skin transparency or the presence of subcutaneous fat obstructing the view, you may euthanize the animal while the skin remains orange/red and excise the section of skin showing the highest dye absorption. The peritoneal membrane may sometimes stick to the skin but can be easily removed. Holding the excised piece of skin up to a light source should reveal its transparency. This also allows you to observe the underlying structures to assess whether they appear red (indicating the depth of dye penetration) and what features (fat, which is usually featureless, vs abdominal organs) are expected to become visible once full skin transparency is achieved.

3.5. Reversal of skin transparency. After achieving skin transparency, thoroughly remove any residual dye from the skin using warm water or warm saline. You may need to apply cotton gauze soaked in warm water or saline to the skin multiple times to fully remove any remaining dye molecules. The skin should return to its normal appearance after rinsing. Leaving these molecules in the skin for too long may cause skin inflammation the next day. To prevent it, always cover the treated area with Neosporin or Eucerin Advanced Repair Cream after rinsing and reversing the skin transparency. Check back after the animal has awakened from anesthesia to apply more Neosporin or Eucerin Advanced Repair Cream as needed, since the animal tends to abrade and remove it when moving actively. One suggested way to prevent the animal from removing the cream during movement is to remove the normal bedding, such as wood chips, from the cage (i.e., use an empty cage for recovery) and place the cage on top of a heating pad for thermal regulation.