f-ORG technique detects early eye disease through light-response analysis

Eye diseases often develop asymptomatically for many years. ICTER scientists have developed the f-ORG technique, which analyzes the retina’s reaction to light, helping to detect danger before the first symptoms appear. New research proves that even the smallest changes in photoreceptors can be detected this way.

The retina is an extremely complex structure that acts as a biological “transducer” of light into neural signals. It is here, in the layer of photoreceptors—cones and rods—that the process of vision begins. Light hitting the outer segments of these cells initiates a series of biochemical reactions known as phototransduction. During this process, the length of the photoreceptors changes, and these microscopic changes—invisible to the naked eye—carry information about the health of the retina.

Previously developed diagnostic methods, such as electroretinography (ERG), have enabled the assessment of photoreceptor function, but they have many limitations. They require contact with the eye surface, long-term adaptation to darkness, and complicated procedures. They are also uncomfortable for patients, especially children and the elderly.

Scientists from the International Centre for Eye Research (ICTER) set out to find a way to overcome these limitations. They developed an innovative technique called “flicker optoretinography” (f-ORG), which facilitates fast, non-invasive, and precise monitoring of the processes occurring in the photoreceptors. This method could revolutionize the diagnosis of retinal diseases, such as macular degeneration, retinitis pigmentosa, and congenital retinal dystrophies.

The results are published in the journal Proceedings of the National Academy of Sciences in an article titled, “Photopic flicker optoretinography captures the light-driven length modulation of photoreceptors during phototransduction.”

“Our method enables tracking of molecular mechanisms of phototransduction without the need for prolonged exposure to darkness and without contact with the surface of the eye. This is a significant step forward in the diagnosis of retinal diseases,” explains Professor Maciej Wojtkowski, co-author of the study.

‘Ultrasound’ for photoreceptors

Flicker electroretinography (f-ERG) is a valuable and successfully used tool for studying the physiological functions of the retina. However, the f-ERG method has its shortcomings, so ICTER scientists decided to develop its optical equivalent. Flicker optoretinography (f-ORG) is a technology that allows for real-time observation of physical changes occurring in the outer segments of the eye’s photoreceptors. These molecular-level elongations are the result of conformational changes in the phosphodiesterase 6 (PDE6) protein.

The cone and rod photoreceptors are extremely sensitive cells that respond to light by lengthening or shortening their outer segments (OS). These changes in OS length reflect the functional activity and health of the retina. The f-ORG technique can record these molecular-level phenomena by using spatial-temporal optical tomography OCT (STOC-T), which enables precise imaging of the retinal architecture in the nanometer range.

The current publication represents another advancement for the ICTER team focusing on f-ORG. In 2022, Prof. Wojtkowski’s research group showed that it is possible to perform f-ORG measurements over a wide frequency range (up to 50 Hz), and in 2024, they proposed a new approach to f-ORG measurements that enables the rapid determination of the frequency characteristics of photoreceptors.

“STOC-T is a real breakthrough. Thanks to it, we can non-invasively track how individual photoreceptors react to light. It’s like having a microscope that works directly in the patient’s eye,” exclaims Andrea Curatolo, Ph.D.

Why is PDE6 so important?

Phosphodiesterase 6 (PDE6) is a key enzyme in phototransduction, the process by which light is converted into electrical signals that the brain interprets as images. PDE6 is located in the outer segments of the rod and cone photoreceptors, and acts as a regulator of the light signal. Its task is to break down cGMP (cyclic guanosine monophosphate), which keeps ion channels open in the dark, allowing sodium and calcium ions to flow into the cell.

When light reaches the retina, the rhodopsin signaling pathway is activated in the photoreceptors, resulting in stimulation of PDE6. This enzyme rapidly breaks down cGMP, causing the ion channels to close and the flow of ions to decrease. The resulting change in electrical potential of the photoreceptor cells is the first step in transmitting visual information to the brain.

“PDE6 is a molecular switch that regulates the sensitivity of photoreceptors to light. Its activation is like pressing the brake pedal in a car—light is the stimulus that starts this process, and PDE6 decides how strong the response will be,” explains Sławomir Tomczewski, Ph.D. Eng, the primary author of the study.

Phototransduction is a process that lasts only fractions of a second at a time, but our ability to see depends on its ability to recycle repetitively in a precise manner. Disturbances in the functioning of the PDE6 regulatory enzyme are associated with many retinal diseases, including retinitis pigmentosa and retinal dystrophies. The new f-ORG technique enables direct observation of the effects of this enzyme’s action in real-time, which gives scientists and doctors a new tool for studying the onset and progression of retinal diseases, and assessing the effectiveness of drug treatments and gene therapies.

How were the studies conducted?

After establishing its safety in preclinical studies, the f-ORG technique was tested on a group of healthy volunteers to confirm its effectiveness in tracking dynamic changes in the photoreceptors and to examine the role of the PDE6 protein in this process. The participants underwent a short, one-minute adaptation to light, which is a significant difference compared to traditional methods requiring a long stay in the dark. Then, their retinas were stimulated with light of a variable frequency—from 1.5 Hz to 45 Hz—and changes in the length of the photoreceptor outer segments were recorded.

The STOC-T technique, performing about 200 three-dimensional scans per second, allowed for the observation of subtle oscillatory extensions of these structures under the influence of light. The measurements showed that the observed extension of the photoreceptors is consistent with theoretical predictions regarding activation of the phototransduction cascade.

In the next stage of the experiments, sildenafil, a PDE6 inhibitor known for its blocking effect on the phototransduction process, was administered to test its effect on the photoreceptor response. As predicted, a significant weakening of the photoreceptor response was observed, which confirmed the key role of PDE6 in the mechanism of elongation of the outer segments of the photosensitive cells.

“This was a breakthrough moment. After the administration of sildenafil, the photoreceptor response decreased significantly. The obtained results seem to confirm that it is indeed the conformational changes in the PDE6 protein that are responsible for the elongation of the outer segments under the influence of light,” says Tomczewski.

What can f-ORG be useful for?

Retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, or congenital dystrophies, often develop unnoticed for many years. Their early diagnosis is extremely difficult because the first clinical symptoms appear when a substantial number of the photoreceptors are already irreversibly damaged.

Current diagnostic methods have focused on visual observation of structural changes and measurements of electrical activity of the retina, omitting subtle structural changes at the molecular level. F-ORG fills this gap, allowing for the recording of changes in the length of the outer segments of photoreceptors, which is a direct indicator of the processes occurring in the retina during light reception.

“Thanks to f-ORG, we can observe the reactions of the retina in real-time. It is like monitoring the operation of an engine without having to dismantle it,” explains Prof. Wojtkowski.

The potential applications of the f-ORG technique are extensive. Owing to to the ability to record the reactions of photoreceptors on a nanoscale, doctors will be able to detect pathological changes much earlier than in the case of traditional methods. The new technique can be used not only in ophthalmology but also in neurology, and for research on neurodegeneration. The retina is a natural “window” to the nervous system and can provide valuable information on the functioning of the brain.

“The f-ORG technique allows us to understand the mechanisms of photoreceptor function, and in the future, it may help find the sources of neurodegenerative diseases at a level not seen before in ophthalmological research,” proclaims Tomczewski.

The future of f-ORG in clinical practice

ICTER scientists plan to further develop the f-ORG technology and adapt it for clinical applications. Preparations are currently underway for studies on patients with early symptoms of macular degeneration and retinitis pigmentosa. There are also plans to develop a portable version of the device that could be used in ophthalmologists’ offices, and even for screening tests of populations at increased risk of retinal diseases.

“We want f-ORG to become a standard in ophthalmology. This is a technology that can help millions of patients around the world by enabling early detection of diseases and more effective treatment,” says Prof. Wojtkowski.

Authors of the paper include Sławomir Tomczewski, Andrea Curatolo, Andrzej Foik, Piotr Węgrzyn, Bartłomiej Bałamut, Maciej Wielgo, Wiktor Kulesza, Anna Galińska, Katarzyna Kordecka, Sahil Gulatie, Humberto Fernandes, Krzysztof Palczewski, Maciej Wojtkowski.