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Finding Better Ways to Image the Retina

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Two light microscopy fields of the retina showing small blue dots (rods) surrounding larger yellow dots (cones)
Credit: Johnny Tam, National Eye Institute, NIH

Every day, all around the world, eye care professionals are busy performing dilated eye exams. By looking through a patient’s widened pupil, they can view the retina—the postage stamp-sized tissue lining the back of the inner eye—and look for irregularities that may signal the development of vision loss.

The great news is that, thanks to research, retinal imaging just keeps getting better and better. The images above, which show the same cells viewed with two different microscopic techniques, provide good examples of how tweaking existing approaches can significantly improve our ability to visualize the retina’s two types of light-sensitive neurons: rod and cone cells.

Specifically, these images show an area of the outer retina, which is the part of the tissue that’s observed during a dilated eye exam. Thanks to colorization and other techniques, a viewer can readily distinguish between the light-sensing, color-detecting cone cells (orange) and the much smaller, lowlight-sensing rod cells (blue).

These high-res images comes from Johnny Tam, a researcher with NIH’s National Eye Institute. Working with Alfredo Dubra, Stanford University, Palo Alto, CA, Tam and his team figured out how to limit light distortion of the rod cells. The key was illuminating the eye using less light, provided as a halo instead of the usual solid, circular beam.

But the researchers’ solution hit a temporary snag when the halo reflected from the rods and cones created another undesirable ring of light. To block it out, Tam’s team introduced a tiny pinhole, called a sub-Airy disk. Along with use of adaptive optics technology [1] to correct for other distortions of light, the scientists were excited to see such a clear view of individual rods and cones. They published their findings recently in the journal Optica [2]

The resolution produced using these techniques is so much improved (33 percent better than with current methods) that it’s even possible to visualize the tiny inner segments of both rods and cones. In the cones, for example, these inner segments help direct light coming into the eye to other, photosensitive parts that absorb single photons of light. The light is then converted into electrical signals that stream to the brain’s visual centers in the occipital cortex, which makes it possible for us to experience vision.

Tam and team are currently working with physician-scientists in the NIH Clinical Center to image the retinas of people with a variety of retinal diseases, including age-related macular degeneration (AMD), a leading cause of vision loss in older adults. These research studies are ongoing, but offer hopeful possibilities for safe and non-intrusive monitoring of individual rods and cones over time, as well as across disease types. That’s obviously good news for patients. Plus it will help scientists understand how a rod or cone cell stops working, as well as more precisely test the effects of gene therapy and other experimental treatments aimed at restoring vision.


[1] Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope. Dubra A, Sulai Y, Norris JL, Cooper RF, Dubis AM, Williams DR, Carroll J. Biomed Opt Express. 2011 Jul 1;2(7):1864-76.

[1] In-vivo sub-diffraction adaptive optics imaging of photoreceptors in the human eye with annular pupil illumination and sub-Airy detection. Rongwen L, Aguilera N, Liu T, Liu J, Giannini JP, Li J, Bower AJ, Dubra A, Tam J. Optica 2021 8, 333-343.


Get a Dilated Eye Exam (National Eye Institute/NIH)

How the Eyes Work (NEI)

Eye Health Data and Statistics (NEI)

Tam Lab (NEI)

Dubra Lab (Stanford University, Palo Alto, CA)

NIH Support: National Eye Institute

Nano-Sized Solution for Efficient and Versatile CRISPR Gene Editing

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Nanocapsule for Gene Editing
Caption: Nanocapsule containing its gene-editing payload.
Credit: Guojun Chen and Amr Abdeen, University of Wisconsin-Madison

If used to make non-heritable genetic changes, CRISPR gene-editing technology holds tremendous promise for treating or curing a wide range of devastating disorders, including sickle cell disease, vision loss, and muscular dystrophy. Early efforts to deliver CRISPR-based therapies to affected tissues in a patient’s body typically have involved packing the gene-editing tools into viral vectors, which may cause unwanted immune reactions and other adverse effects.

Now, NIH-supported researchers have developed an alternative CRISPR delivery system: nanocapsules. Not only do these tiny, synthetic capsules appear to pose a lower risk of side effects, they can be precisely customized to deliver their gene-editing payloads to many different types of cells or tissues in the body, which can be extremely tough to do with a virus. Another advantage of these gene-editing nanocapsules is that they can be freeze-dried into a powder that’s easier than viral systems to transport, store, and administer at different doses.

In findings published in Nature Nanotechnology [1], researchers, led by Shaoqin Gong and Krishanu Saha, University of Wisconsin-Madison, developed the nanocapsules with specific design criteria in mind. They would need to be extremely small, about the size of a small virus, for easy entry into cells. Their surface would need to be adaptable for targeting different cell types. They also had to be highly stable in the bloodstream and yet easily degraded to release their contents once inside a cell.

After much hard work in the lab, they created their prototype. It features a thin polymer shell that’s easily decorated with peptides or other ingredients to target the nanocapsule to a predetermined cell type.

At just 25 nanometers in diameter, each nanocapsule still has room to carry cargo. That cargo includes a single CRISPR/Cas9 scissor-like enzyme for snipping DNA and a guide RNA that directs it to the right spot in the genome for editing.

In the bloodstream, the nanocapsules remain fully intact. But, once inside a cell, their polymer shells quickly disintegrate and release the gene-editing payload. How is this possible? The crosslinking molecules that hold the polymer together immediately degrade in the presence of another molecule, called glutathione, which is found at high levels inside cells.

The studies showed that human cells grown in the lab readily engulf and take the gene-editing nanocapsules into bubble-like endosomes. Their gene-editing contents are then released into the cytoplasm where they can begin making their way to a cell’s nucleus within a few hours.

Further study in lab dishes showed that nanocapsule delivery of CRISPR led to precise gene editing of up to about 80 percent of human cells with little sign of toxicity. The gene-editing nanocapsules also retained their potency even after they were freeze-dried and reconstituted.

But would the nanocapsules work in a living system? To find out, the researchers turned to mice, targeting their nanocapsules to skeletal muscle and tissue in the retina at the back of eye. Their studies showed that nanocapsules injected into muscle or the tight subretinal space led to efficient gene editing. In the eye, the nanocapsules worked especially well in editing retinal cells when they were decorated with a chemical ingredient known to bind an important retinal protein.  

Based on their initial results, the researchers anticipate that their delivery system could reach most cells and tissues for virtually any gene-editing application. In fact, they are now exploring the potential of their nanocapsules for editing genes within brain tissue.

I’m also pleased to note that Gong and Saha’s team is part of a nationwide consortium on genome editing supported by NIH’s recently launched Somatic Cell Genome Editing program. This program is dedicated to translating breakthroughs in gene editing into treatments for as many genetic diseases as possible. So, we can all look forward to many more advances like this one.


[1] A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing. Chen G, Abdeen AA, Wang Y, Shahi PK, Robertson S, Xie R, Suzuki M, Pattnaik BR, Saha K, Gong S. Nat Nanotechnol. 2019 Sep 9.


Somatic Cell Genome Editing (NIH)

Saha Lab (University of Wisconsin-Madison)

Shaoqin (Sarah) Gong (University of Wisconsin-Madison)

NIH Support: National Eye Institute; National Institute of General Medical Sciences; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; Common Fund

The Amazing Brain: Making Up for Lost Vision

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Recently, I’ve highlighted just a few of the many amazing advances coming out of the NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative. And for our grand finale, I’d like to share a cool video that reveals how this revolutionary effort to map the human brain is opening up potential plans to help people with disabilities, such as vision loss, that were once unimaginable.

This video, produced by Jordi Chanovas and narrated by Stephen Macknik, State University of New York Downstate Health Sciences University, Brooklyn, outlines a new strategy aimed at restoring loss of central vision in people with age-related macular degeneration (AMD), a leading cause of vision loss among people age 50 and older. The researchers’ ultimate goal is to give such people the ability to see the faces of their loved ones or possibly even read again.

In the innovative approach you see here, neuroscientists aren’t even trying to repair the part of the eye destroyed by AMD: the light-sensitive retina. Instead, they are attempting to recreate the light-recording function of the retina within the brain itself.

How is that possible? Normally, the retina streams visual information continuously to the brain’s primary visual cortex, which receives the information and processes it into the vision that allows you to read these words. In folks with AMD-related vision loss, even though many cells in the center of the retina have stopped streaming, the primary visual cortex remains fully functional to receive and process visual information.

About five years ago, Macknik and his collaborator Susana Martinez-Conde, also at Downstate, wondered whether it might be possible to circumvent the eyes and stream an alternative source of visual information to the brain’s primary visual cortex, thereby restoring vision in people with AMD. They sketched out some possibilities and settled on an innovative system that they call OBServ.

Among the vital components of this experimental system are tiny, implantable neuro-prosthetic recording devices. Created in the Macknik and Martinez-Conde labs, this 1-centimeter device is powered by induction coils similar to those in the cochlear implants used to help people with profound hearing loss. The researchers propose to surgically implant two of these devices in the rear of the brain, where they will orchestrate the visual process.

For technical reasons, the restoration of central vision will likely be partial, with the window of vision spanning only about the size of one-third of an adult thumbnail held at arm’s length. But researchers think that would be enough central vision for people with AMD to regain some of their lost independence.

As demonstrated in this video from the BRAIN Initiative’s “Show Us Your Brain!” contest, here’s how researchers envision the system would ultimately work:

• A person with vision loss puts on a specially designed set of glasses. Each lens contains two cameras: one to record visual information in the person’s field of vision; the other to track that person’s eye movements enabled by residual peripheral vision.
• The eyeglass cameras wirelessly stream the visual information they have recorded to two neuro-prosthetic devices implanted in the rear of the brain.
• The neuro-prosthetic devices process and project this information onto a specific set of excitatory neurons in the brain’s hard-wired visual pathway. Researchers have previously used genetic engineering to turn these neurons into surrogate photoreceptor cells, which function much like those in the eye’s retina.
• The surrogate photoreceptor cells in the brain relay visual information to the primary visual cortex for processing.
• All the while, the neuro-prosthetic devices perform quality control of the visual signals, calibrating them to optimize their contrast and clarity.

While this might sound like the stuff of science-fiction (and this actual application still lies several years in the future), the OBServ project is now actually conceivable thanks to decades of advances in the fields of neuroscience, vision, bioengineering, and bioinformatics research. All this hard work has made the primary visual cortex, with its switchboard-like wiring system, among the brain’s best-understood regions.

OBServ also has implications that extend far beyond vision loss. This project provides hope that once other parts of the brain are fully mapped, it may be possible to design equally innovative systems to help make life easier for people with other disabilities and conditions.


Age-Related Macular Degeneration (National Eye Institute/NIH)

Macknik Lab (SUNY Downstate Health Sciences University, Brooklyn)

Martinez-Conde Laboratory (SUNY Downstate Health Sciences University)

Show Us Your Brain! (BRAIN Initiative/NIH)

Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative (NIH)

NIH Support: BRAIN Initiative

‘Nanoantennae’ Make Infrared Vision Possible

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Nanoparticles for infrared vision
Caption: Nanoparticles (green) bind to light-sensing rod (violet) and cone (red) cells in the mouse retina. Dashed lines (white) highlight cells’ inner/outer segments.
Credit: Ma et al. Cell, 2019

Infrared vision often brings to mind night-vision goggles that allow soldiers to see in the dark, like you might have seen in the movie Zero Dark Thirty. But those bulky goggles may not be needed one day to scope out enemy territory or just the usual things that go bump in the night. In a dramatic advance that brings together material science and the mammalian vision system, researchers have just shown that specialized lab-made nanoparticles applied to the retina, the thin tissue lining the back of the eye, can extend natural vision to see in infrared light.

The researchers showed in mouse studies that their specially crafted nanoparticles bind to the retina’s light-sensing cells, where they act like “nanoantennae” for the animals to see and recognize shapes in infrared—day or night—for at least 10 weeks. Even better, the mice maintained their normal vision the whole time and showed no adverse health effects. In fact, some of the mice are still alive and well in the lab, although their ability to see in infrared may have worn off.

When light enters the eyes of mice, humans, or any mammal, light-sensing cells in the retina absorb wavelengths within the range of visible light. (That’s roughly from 400 to 700 nanometers.) While visible light includes all the colors of the rainbow, it actually accounts for only a fraction of the full electromagnetic spectrum. Left out are the longer wavelengths of infrared light. That makes infrared light invisible to the naked eye.

In the study reported in the journal Cell, an international research team including Gang Han, University of Massachusetts Medical School, Worcester, wanted to find a way for mammalian light-sensing cells to absorb and respond to the longer wavelengths of infrared [1]. It turns out Han’s team had just the thing to do it.

His NIH-funded team was already working on the nanoparticles now under study for application in a field called optogenetics—the use of light to control living brain cells [2]. Optogenetics normally involves the stimulation of genetically modified brain cells with blue light. The trouble is that blue light doesn’t penetrate brain tissue well.

That’s where Han’s so-called upconversion nanoparticles (UCNPs) came in. They attempt to get around the normal limitations of optogenetic tools by incorporating certain rare earth metals. Those metals have a natural ability to absorb lower energy infrared light and convert it into higher energy visible light (hence the term upconversion).

But could those UCNPs also serve as miniature antennae in the eye, receiving infrared light and emitting readily detected visible light? To find out in mouse studies, the researchers injected a dilute solution containing UCNPs into the back of eye. Such sub-retinal injections are used routinely by ophthalmologists to treat people with various eye problems.

These UCNPs were modified with a protein that allowed them to stick to light-sensing cells. Because of the way that UCNPs absorb and emit wavelengths of light energy, they should to stick to the light-sensing cells and make otherwise invisible infrared light visible as green light.

Their hunch proved correct, as mice treated with the UCNP solution began seeing in infrared! How could the researchers tell? First, they shined infrared light into the eyes of the mice. Their pupils constricted in response just as they would with visible light. Then the treated mice aced a series of maneuvers in the dark that their untreated counterparts couldn’t manage. The treated animals also could rely on infrared signals to make out shapes.

The research is not only fascinating, but its findings may also have a wide range of intriguing applications. One could imagine taking advantage of the technology for use in hiding encrypted messages in infrared or enabling people to acquire a temporary, built-in ability to see in complete darkness.

With some tweaks and continued research to confirm the safety of these nanoparticles, the system might also find use in medicine. For instance, the nanoparticles could potentially improve vision in those who can’t see certain colors. While such infrared vision technologies will take time to become more widely available, it’s a great example of how one area of science can cross-fertilize another.


[1] Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae. Ma Y, Bao J, Zhang Y, Li Z, Zhou X, Wan C, Huang L, Zhao Y, Han G, Xue T. Cell. 2019 Feb 27. [Epub ahead of print]

[2] Near-Infrared-Light Activatable Nanoparticles for Deep-Tissue-Penetrating Wireless Optogenetics. Yu N, Huang L, Zhou Y, Xue T, Chen Z, Han G. Adv Healthc Mater. 2019 Jan 11:e1801132.


Diagram of the Eye (National Eye Institute/NIH)

Infrared Waves (NASA)

Visible Light (NASA)

Han Lab (University of Massachusetts, Worcester)

NIH Support: National Institute of Mental Health; National Institute of General Medical Sciences

Moving Closer to a Stem Cell-Based Treatment for AMD

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In recent years, researchers have figured out how to take a person’s skin or blood cells and turn them into induced pluripotent stem cells (iPSCs) that offer tremendous potential for regenerative medicine. Still, it’s been a challenge to devise safe and effective ways to move this discovery from the lab into the clinic. That’s why I’m pleased to highlight progress toward using iPSC technology to treat a major cause of vision loss: age-related macular degeneration (AMD).

In the new work, researchers from NIH’s National Eye Institute developed iPSCs from blood-forming stem cells isolated from blood donated by people with advanced AMD [1]. Next, these iPSCs were exposed to a variety of growth factors and placed on supportive scaffold that encouraged them to develop into healthy retinal pigment epithelium (RPE) tissue, which nurtures the light-sensing cells in the eye’s retina. The researchers went on to show that their lab-grown RPE patch could be transplanted safely into animal models of AMD, preventing blindness in the animals.

This preclinical work will now serve as the foundation for a safety trial of iPSC-derived RPE transplants in 12 human volunteers who have already suffered vision loss due to the more common “dry” form of AMD, for which there is currently no approved treatment. If all goes well, the NIH-led trial may begin enrolling patients as soon as this year.

Risk factors for AMD include a combination of genetic and environmental factors, including age and smoking. Currently, more than 2 million Americans have vision-threatening AMD, with millions more having early signs of the disease [2].

AMD involves progressive damage to the macula, an area of the retina about the size of a pinhead, made up of millions of light-sensing cells that generate our sharp, central vision. Though the exact causes of AMD are unknown, RPE cells early on become inflamed and lose their ability to clear away debris from the retina. This leads to more inflammation and progressive cell death.

As RPE cells are lost during the “dry” phase of the disease, light-sensing cells in the macula also start to die and reduce central vision. In some people, abnormal, leaky blood vessels will form near the macula, called “wet” AMD, spilling fluid and blood under the retina and causing significant vision loss. “Wet” AMD has approved treatments. “Dry” AMD does not.

But, advances in iPSC technology have brought hope that it might one day be possible to shore up degenerating RPE in those with dry AMD, halting the death of light-sensing cells and vision loss. In fact, preliminary studies conducted in Japan explored ways to deliver replacement RPE to the retina [3]. Though progress was made, those studies highlighted the need for more reliable ways to produce replacement RPE from a patient’s own cells. The Japanese program also raised concerns that iPSCs derived from people with AMD might be prone to cancer-causing genomic changes.

With these challenges in mind, the NEI team led by Kapil Bharti and Ruchi Sharma have designed a more robust process to produce RPE tissue suitable for testing in people. As described in Science Translational Medicine, they’ve come up with a three-step process.

Rather than using fibroblast cells from skin as others had done, Bharti and Sharma’s team started with blood-forming stem cells from three AMD patients. They reprogrammed those cells into “banks” of iPSCs containing multiple different clones, carefully screening them to ensure that they were free of potentially cancer-causing changes.

Next, those iPSCs were exposed to a special blend of growth factors to transform them into RPE tissue. That recipe has been pursued by other groups for a while, but needed to be particularly precise for this human application. In order for the tissue to function properly in the retina, the cells must assemble into a uniform sheet, just one-cell thick, and align facing in the same direction.

So, the researchers developed a specially designed scaffold made of biodegradable polymer nanofibers. That scaffold helps to ensure that the cells orient themselves correctly, while also lending strength for surgical transplantation. By spreading a single layer of iPSC-derived RPE progenitors onto their scaffolds and treating it with just the right growth factors, the researchers showed they could produce an RPE patch ready for the clinic in about 10 weeks.

To test the viability of the RPE patch, the researchers first transplanted a tiny version (containing about 2,500 RPE cells) into the eyes of a rat with a compromised immune system, which enables human cells to survive. By 10 weeks after surgery, the human replacement tissue had integrated into the animals’ retinas with no signs of toxicity.

Next, the researchers tested a larger RPE patch (containing 70,000 cells) in pigs with an AMD-like condition. This patch is the same size the researchers ultimately would expect to use in people. Ten weeks after surgery, the RPE patch had integrated into the animals’ eyes, where it protected the light-sensing cells that are so critical for vision, preventing blindness.

These results provide encouraging evidence that the iPSC approach to treating dry AMD should be both safe and effective. But only a well-designed human clinical trial, with all the appropriate prior oversights to be sure the benefits justify the risks, will prove whether or not this bold approach might be the solution to blindness faced by millions of people in the future.

As the U.S. population ages, the number of people with advanced AMD is expected to rise. With continued progress in treatment and prevention, including iPSC technology and many other promising approaches, the hope is that more people with AMD will retain healthy vision for a lifetime.


[1] Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sharma R, Khristov V, Rising A, Jha BS, Dejene R, Hotaling N, Li Y, Stoddard J, Stankewicz C, Wan Q, Zhang C, Campos MM, Miyagishima KJ, McGaughey D, Villasmil R, Mattapallil M, Stanzel B, Qian H, Wong W, Chase L, Charles S, McGill T, Miller S, Maminishkis A, Amaral J, Bharti K. Sci Transl Med. 2019 Jan 16;11(475).

[2] Age-Related Macular Degeneration, National Eye Institute.

[3] Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration. Mandai M, Watanabe A, Kurimoto Y, Hirami Y, Takasu N, Ogawa S, Yamanaka S, Takahashi M, et al. N Engl J Med. 2017 Mar 16;376(11):1038-1046.


Facts About Age-Related Macular Degeneration (National Eye Institute/NIH)

Stem Cell-Based Treatment Used to Prevent Blindness in Animal Models of Retinal Degeneration (National Eye Institute/NIH)

Kapil Bharti (NEI)

NIH Support: National Eye Institute; Common Fund

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