Looking Sharply into the Eyes

Innovative imaging technology developed at Technion’s Dept. of Biomedical Engineering captures fine, high quality optical images of retinal structures in vivo. The system, which can be easily integrated into any existing two-photon microscope without requiring adaptive corrections, could potentially be transformative for retina research.

Prof. Shy Shoham

Prof. Shy Shoham, Technion

The recent innovation, described in a new publication in Nature Publishing‘s journal Light: Science & Applications, enables non-invasive two-photon imaging of mouse retinas in vivo, and could therefore potentially transform eye research and our understanding of the mechanisms that underlie retinal physiology. The study describes an optical system based on add-on optics that can be easily integrated into essentially any existing two-photon microscope, and includes an electronically tunable lens for motionless scanning of the depth dimension. The system’s simplicity effectively opens up a new range of potential applications for two-photon excitation microscopy – an advanced fluorescence imaging technique already installed today in numerous laboratories. This new imaging technology was developed at the Neural Interface Engineering Laboratory in the Technion’s Faculty of Biomedical Engineering by doctoral student Adi Schejter Bar-Noam, the laboratory head, Prof. Shy Shoham, and research associate Dr. Nairouz Farah.

Two-photon microscopy provides fluorescence imaging with sub-cellular resolution, contributing to the understanding of cells and multicellular structures in biological tissues. The key advantage of this approach is its ability to penetrate relatively deeply into tissues, creating three-dimensional images consisting of a series of two-dimensional cross-sections; this is what turned it in the last two decades into a principal tool in studies focusing on the structure and operation of the brain and nervous system. However, to date, it was not harnessed for in vivo retinal imaging due to focusing constraints arising from the combination of common two-photon imaging lenses and the optics of the mouse eye.

Now, thanks to this novel imaging technology, it is possible to visualize entire regions in the retina, including blood vessels, nerve cells and more – at high resolutions and non-invasively. Since the system uses near-infrared (NIR) light-based two-photon excitation, the microscopic examination does not strongly impact the behavior of the imaged retina and allows it to “see” almost normally during imaging. The researchers used this in order to directly measure in vivo natural neural responses to flashes of light for the first time.


Doctoral student Adi Schejter Bar-Noam

“Our motivation in developing this new technology was for research purposes – to improve the methods available to us as scientists,” explains Schejter Bar-Noam who developed the system. “Perhaps in the future, the new system will accommodate the development of translational vision applications, although our current study was limited to researching the retina and its neurons. The system provides an advanced in vivo microscopic image of the living retina without causing it any damage; we confirmed that prolonged use of the system did not cause any damage to the examined retina.”

“The broader context of our work is Optogenetics,” adds Prof. Shy Shoham, “a scientific area that has flourished over the past decade and allows us to study neurons over time using fluorescent proteins, or to use light-sensitive proteins to activate cells. The new system allows us to make use of the two-photon microscope in both aspects, that is, in the imaging of retinal neurons or for cellular activation by light. Indeed, the extended design process demonstrated its effectiveness for imaging nerve cells and blood vessel.”

The research was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program, by the Israel Science Foundation (ISF), and by a generous donation by the Cohen-CTS fund.

Green Scale Demo

3D imaging of blood vessels around the optic nerve in the retina. This is a 3D reconstruction of a few “cuts” in the retina depth. The blood vessels were colored with Fluorescein. The length of the white line is 100 micron