Laser Ophthalmoscopic Method Enables In Vivo Imaging of the Eye

An instrument called the two-photon excited fluorescence scanning laser ophthalmoscope (TPEF-SLO) has made it possible to view, in real time, the biochemical processes that occur in the retina. Researchers have traditionally been unable to view these processes.

Developed by a team led by professor Maciej Wojtkowski from the International Centre for Translational Eye Research (ICTER), the TPEF-SLO is a compact instrument that provides spectrally resolved images of the human retina based on two-photon excitation (TPE) with near-infrared (NIR) light. A custom fiber laser with integrated pulse selection, along with intelligent post-processing of data, enables excitation with low laser power and precise measurement of weak signals.

TPEF-SLO allows users to noninvasively assess the metabolic processes that support the regeneration of human retinal visual pigments (i.e., the visual cycle). Users can observe, in vivo, the molecules that are present in the environment of retinal photosensitive cells. The real-time observation of these molecules, which sustain visual function, could enable clinicians to monitor eye diseases in the earliest stages, before structural damage to the retina occurs.

“The human eye is a biochemical factory whose activity depends on biochemical transformations of a single molecule, retinal. This molecule is indispensable for the function of the visual pigments, namely rhodopsin in rods,” Wojtkowski said.

The retinal molecules that support rhodopsin, a light-sensitive pigment needed for vision in dim light, cannot be detected by existing scientific instruments during virtually the entire visual cycle in living humans. “However, there is one instant in the visual cycle when the molecules can be seen; we can’t detect them with UV light, but we can observe them thanks to so-called fluorescence with two-photon excitation,” researcher Jakub Boguslawski said.

TPE is an advanced technique for measuring compounds that support the function of visual pigments; these compounds are not visible through other testing techniques. TPE makes it possible to view fluorescent vitamin A derivatives — chemical intermediates of biochemical processes in the eye that are highly sensitive to UV light. Eye tissue such as the sclera, cornea, and lens are highly transparent to NIR light, which penetrates retinal tissues in a noninvasive way.

In age-related macular degeneration, cells within a disease-altered retina cannot be distinguished at an early stage from cells of a normal healthy retina. However, the differences can be picked up by biochemical markers, if these markers can be fluorescently induced.

Ophthalmic imaging techniques such as OCT and scanning laser ophthalmoscopy (SLO) are essential tools for diagnosing retinal pathologies. They cannot provide a complete view of the eye’s biochemical processes.

Images obtained with TPEF-SLO confirmed its effectiveness as a way to view the molecules that sustain visual function. Comparison of data from humans with retinal degeneration with mouse models of the disease revealed a similar rapid accumulation of bisretinoid condensation products, the researchers said.

“We believe that visual cycle intermediates and toxic byproducts of this metabolic pathway could be measured and quantified using TPE imaging,” researcher Grazyna Palczewska said.

Scientists can see the chemistry of vision through TPEF-SLO, a new instrument and method developed at the International Centre for Translational Eye Research in Poland. Courtesy of the International Centre for Translational Eye Research (ICTER), Grzegorz Krzyzewski.

The development of TPEF-SLO opens numerous therapeutic possibilities for degenerative diseases of the retina, including the testing of new drugs. By understanding the biochemistry of vision and the alterations that occur in disease, physicians will be able to pinpoint precise locations of the lesions and assess the impact of therapy.

“Thanks to close collaborations with biochemist professor Kris Palczewski from the University of California, Irvine and the laser group of professor Grzegorz Sobon from the Wroclaw University of Science and Technology, we can quickly and effectively demonstrate the capabilities of the new imaging method and validate its utility for diagnosing disease progression and treatment, leading to its use in clinical practice,” Wojtkowski said.

The research was published in The Journal of Clinical Investigation (www.jci.org/articles/view/154218#top).