New technique makes skin invisible

Researchers have developed a new method to visualize a body’s organs by making the overlying tissues transparent to visible light. The counterintuitive process—a topical application of food coloring—was shown to be reversible in animal tests and could be applied to a wide range of medical diagnostics, from locating injuries to monitoring digestive disorders to identifying cancers.

Researchers at Stanford University published the research, titled “Achieving Optical Transparency in Living Animals with Absorbing Molecules,” in the September 6, 2024, issue of Science.

“In the future, this technology could make veins more visible for blood sampling, simplify laser tattoo removal or aid in the early detection and treatment of cancers,” said Guosong Hong, an assistant professor of materials science and engineering at Stanford University who helped lead the work.

“For example, some therapies use lasers to eliminate cancerous and precancerous cells, but are limited to areas close to the surface of the skin. This technique could improve the penetration of light.”

An enlightening solution

To master the new technique, the researchers developed a way to predict how light interacts with colored biological tissues.

These predictions required a deep understanding of light scattering, as well as the process of refraction, where light changes speed and bends as it travels from one material to another.

Scattering is the reason we can’t see through our bodies: fats, fluids in cells, proteins, and other materials each have a different refractive index, a property that determines how much an incoming light wave will bend.

In most tissues, these materials are tightly packed, so that the different refractive indices cause light to scatter as it passes through them. It is this scattering effect that our eyes interpret as opaque, colored biological materials.

The researchers realized that if they wanted to make the biological material transparent, they needed to find a way to match the different refractive indices so that light could pass through unimpeded.

Drawing on fundamental knowledge from the field of optics, the researchers realized that the dyes most efficient at absorbing light can also be very efficient at directing light uniformly across a wide range of refractive indices.

The researchers predicted that one dye would be particularly effective: tartrazine, the food coloring better known as FD&C Yellow 5. It turns out they were right: When dissolved in water and absorbed by tissues, tartrazine molecules are perfectly structured to match refractive indices and prevent light from scattering, resulting in transparency.

The researchers first tested their predictions with thin slices of chicken breast. As tartrazine concentrations increased, the refractive index of the fluid in the muscle cells increased until it matched the refractive index of the muscle proteins: the slice became transparent.

The researchers then gently applied a temporary solution of tartrazine to the mice. They first applied the solution to the scalp, making the skin transparent to reveal the blood vessels that crisscross the brain. They then applied the solution to the abdomen, which faded within minutes to reveal contractions of the intestine and movements caused by heartbeat and breathing.

The technique allowed the detection of micron-scale features and even improved microscopic observations. Once the dye was removed, the tissues quickly returned to normal opacity. Tartrazine did not appear to have any long-term effects, and any excess was eliminated in the waste within 48 hours.

The researchers suspect that injecting the dye should allow for even deeper observations within organisms, with implications for both biology and medicine.

Old formulas open new window on medicine

The project began with a study of how microwave radiation interacts with biological tissues.

Digging through optics textbooks from the 1970s and 1980s, the researchers discovered two key concepts: mathematical equations called Kramers-Kronig relations and a phenomenon called Lorentz oscillation, where electrons and atoms resonate within molecules as photons pass through them.

Well-studied for more than a century, but never applied to medicine in this way, these tools have proven ideal for predicting how a given dye can increase the refractive index of biological fluids to perfectly match surrounding fats and proteins.

Graduate researcher Nick Rommelfanger, working under an NSF graduate research fellowship, was one of the first to realize that the same modifications that make materials transparent to microwaves could be tailored to impact the visible spectrum, with potential applications in medicine.

A molecule among many others

Moving from theory to experiment, postdoctoral researcher Zihao Ou, lead author of the study, ordered a number of powerful dyes and began the process of meticulously evaluating each one to determine its ideal optical properties.

Eventually, the team grew to 21 students, collaborators and advisors, involving multiple analytical systems.

One item that proved crucial was a decades-old ellipsometer nestled among newer equipment at the Stanford Nano Shared Facility, part of the NSF’s National Nanotechnology Coordinated Infrastructure (NNCI).

The ellipsometer is a tool familiar to semiconductor manufacturing, not biology. However, in what may be a first for medicine, the researchers realized it was perfect for predicting the optical properties of their target dyes.

“Advanced research facilities are constantly seeking to strike the right balance between providing access to core tools and expertise while making room for newer, larger, more powerful instrumentation,” said Richard Nash, NSF program officer who oversees the NSF NNCI.

“While a basic instrument like an ellipsometer rarely makes headlines, it can nevertheless play a crucial role when used for atypical uses as in this case. Open access to this type of instrumentation is fundamental to making revolutionary discoveries, because these instruments can be deployed in new ways to generate fundamental knowledge about scientific phenomena.”

Using methods grounded in fundamental physics, the researchers hope their approach will launch a new field of study matching dyes to biological tissues based on optical properties, which could lead to a wide range of medical applications.

“As an optician, I’m amazed at how much they’ve been able to leverage the Kramers-Konig relationship,” said Adam Wax, an NSF program manager who supported Hong’s work.

“Every optics student knows them, but this team used the equations to understand how a highly absorbing dye can make skin transparent. … Hong was able to take a bold new direction, a great example of how fundamental knowledge of optics can be used to create new technologies, including in biomedicine.”