Let’s clear things up: how do glassfrogs achieve transparency?

Tissue transparency—a useful skill for animals that want to hide from predators—is common in aquatic environments, but is extremely rare on land. One exception is the glassfrog, so named because its internal organs can be seen through its transparent skin and muscles. This frog is active at night and spends its days sleeping on leaves, becoming nearly invisible on the foliage. But how exactly this creature makes itself transparent has been somewhat of a mystery.

Using state-of-the-art imaging technology, NIH-funded researchers have found the secret behind the glassfrog’s camouflage. Their findings, recently published on the cover of Science, demonstrate that glassfrogs can remove almost 90% of their red blood cells from circulation, storing them in the liver during rest. This mechanism, along with its inherently see-through tissues, allows the glassfrog to increase its transparency by two- to threefold, seemingly on demand.

“In vertebrates like humans, tissue transparency is particularly difficult to achieve, as our circulatory systems are filled with oxygen-carrying red blood cells that strongly absorb light and render our tissues opaque,” explained NIBIB-funded researcher Junjie Yao, Ph.D., an assistant professor of biomedical engineering at Duke University. “In our study, we have discovered that the glassfrog can conceal nearly all of its red blood cells within its liver on a daily basis, resulting in a unique form of camouflage that is distinct from all other known mechanisms of tissue transparency. Understanding this blood flow mechanism in the glassfrog may provide insights into disorders related to blood clotting or stroke in humans.”

The glassfrog’s camouflage is adaptive—the creature has peak transparency when it is asleep and at increased risk of attack. To understand the mechanism of this transparency, the researchers first used spectroscopy techniques to passively measure glassfrog’s levels of hemoglobin (a protein in red blood cells that carries oxygen and absorbs light). They found that hemoglobin levels were barely distinguishable when the frogs were sleeping, but markedly increased after exercise. This means that the glassfrog depletes its red blood cells from circulation during rest, allowing for enhanced tissue transparency and camouflage during this vulnerable time.

But where exactly are these red blood cells going while the glassfrog is asleep? To answer this question, the researchers used an advanced technique called photoacoustic microscopy, a hybrid imaging method that takes advantage of both light and sound waves. Here’s how it works: When a tissue absorbs light, some of that absorbed energy is converted into ultrasound waves. Measuring these ultrasound waves with a specialized transducer can give detailed information about molecules and tissues under the skin’s surface. In this study, the researchers optimized this technique so that they could detect light absorbed by hemoglobin—this way, the resulting ultrasound waves would give information about glassfrogs’ red blood cell movement.

“Photoacoustic microscopy allowed us to capture the blood flow dynamics of the glassfrogs, even though those changes occur deep inside their opaque internal organs,” explained Carlos Taboada, Ph.D., a postdoctoral associate at Duke University and one of the leading authors of this study. “This aspect of the technique was really important, because many internal organs of the glassfrog contain millions of nanocrystals that attenuate most incident light. Traditional imaging methods can’t track blood flow with such a high level of accuracy or tell us exactly where the glassfrogs are storing their red blood cells.”

Using photoacoustic microscopy, the researchers imaged the glassfrogs while under anesthesia (which leads to red blood cells being distributed throughout the entire body), while sleeping, and after exercise. During rest, the researchers found that the glassfrogs’ circulating pool of red blood cells decreased by 80-90%, with the hemoglobin signal concentrated in the liver. Imaging the frogs after exercise revealed that the blood flows out of the liver and back into circulation during activity, with the red blood cells reaggregating in the liver within roughly an hour after movement.

By revealing the mechanism of the glassfrog’s camouflage, a new question has surfaced: How do these creatures concentrate most of their red blood cells in their liver without triggering clotting events? “At this point, we really know very little about this important physiological function in glassfrogs, but we are actively working to understand this phenomenon, which has significant clinical implications,” said Yao. “This anti-clotting mechanism is highly relevant for treating thrombosis and stroke, as well as improving medical interventions that require removing blood from the body (like dialysis), where blood clotting is a major concern.”

Future work in this space can take advantage of the unique ability of photoacoustic imaging to track glassfrog’s blood flow—in a safe and unobtrusive way that does not disturb the animals. “By optimizing photoacoustic imaging to specifically detect hemoglobin, the researchers were able to observe the glassfrogs’ natural blood flow dynamics without injecting contrast agents, representing a truly non-invasive approach,” said Randy King, Ph.D., a program director in the Division of Applied Science & Technology at NIBIB. “What’s more, this study highlights how photoacoustic microscopy can be tailored to investigate different aspects of blood dynamics, providing both high-resolution and real-time information.”

This study was supported by grants from NIBIB (R01EB028143), the National Institute of Neurological Disorders and Stroke (NINDS; R01NS111039), and the NIH BRAIN Initiative (RF1NS115581).

This Science Highlight describes a basic research finding. Basic research increases our understanding of human behavior and biology, which is foundational to advancing new and better ways to prevent, diagnose, and treat disease. Science is an unpredictable and incremental process—each research advance builds on past discoveries, often in unexpected ways. Most clinical advances would not be possible without the knowledge of fundamental basic research.

Study reference: Carlos Taboada et al. Glassfrogs conceal blood in their liver to maintain transparency. Science (2022). DOI: 10.1126/science.abl6620