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

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.