Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae

Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper. I picked this months paper because it is just too cool not to talk about! Published just a couple of days ago online, this month’s paper is “Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae” by Xue Tian and colleagues at University of Science & Technology of China. I will provide a brief overview of the techniques/approaches used to make it more understandable to potential non-expert readers. If I can’t figure something out, I’ll just say so.

Have you ever wanted to see like a rattlesnake? Have you ever yearned to have ‘thermal vision’, the type you’ve undoubtedly seen on an average episode of “Cops”? Well, thanks to recent advances in science, you may soon be able to! Our vision is restricted to wavelengths of light falling between 400 and 700 nm…that’s it, everything you’ve ever seen or can ever see is due to your retina interpreting light in this range. This is great, but it is so limiting, so much so that most people never even think about what they are missing in the non-visible range of light. Indeed, things we can see fall into < 1% of the total range of the electromagnetic spectrum (see below; visible + non-visible). Imagine what we could see with 2% of the spectrum covered!

To expand the visual capabilities of mice into the near-infrared (NIR) range, the researchers developed a nanoparticle based ‘nanoantenna’ that is injectable (into the eye), self-powered, and binds normal photoreceptors (rods and cones) in the retina. These retinal photoreceptor-binding up-conversion nanoparticles (pbUCNPs) work as mini-transducers, capable of transforming NIR light into short wavelength (visible) emissions in vivo (that is, in the living animal), that the mouse can then see normally. (Image credits: Steven White, Quora.com; Newpaper24.com)

Anyone who has worked with fluorescent particles (e.g., those conjugated to secondary antibodies), knows about excitation/emission spectra. This reflects the wavelength of light that excites the fluorescent particle (in this case, nanoparticle), and then reciprocally, what type of light the particle gives off (emission). The researchers developed so-called ‘up-conversion’ nanoparticles to allow mice to see NIR light. This means that the emission spectra of these nano-antenna were of smaller (higher energy) wavelengths of light than the excitation spectra. Specifically, these nano-antenna are excited by NIR light (~980 nm wavelength), but give off light in the visible spectrum (~535 nm wavelength). Additional modifications were made to the nanoparticles to make them water-soluble (so they could be injected in phosphate buffered saline; PBS), and make them bind (uniformly) to rods and cones in the retina). Through these biochemical tricks, they were able to create nanoantennae that sense NIR light, respond to that light by emitting light in the visible spectrum, and bind to natural photoreceptors in the retina! As an added bonus, they further showed that these nano-antennae are non-toxic (at least for 2 months), as they did not cause photoreceptor degeneration or marked activation of immune cells within the retina (microglia).

Photoreceptor-binding Up-Conversion NanoParticles (pbUNCPs) bind to natural photoreceptors (rods and cones). Above, you can see that when mice were injected with just PBS, no pbUCNP signal is observed…however when they are injected with PBS + pbUCNPs….the nanoparticles latch onto existing rods and cones, showing that they can ‘hijack’ or ‘co-opt’ normal visual pathways in the retina  (Ma et al., 2019).

Photoreceptor-binding Up-Conversion NanoParticles (pbUNCPs) bind to natural photoreceptors (rods and cones). Above, you can see that when mice were injected with just PBS, no pbUCNP signal is observed…however when they are injected with PBS + pbUCNPs….the nanoparticles latch onto existing rods and cones, showing that they can ‘hijack’ or ‘co-opt’ normal visual pathways in the retina (Ma et al., 2019).

Ok, so how did the researchers tell if the mice could indeed see the NIR light? Their first test was a simple pupillary light reflex (PLR) test. As light intensity increases, our pupils (and those of mice) constrict to prevent damage to our retinas (think about when you got your pupils dilated…and how sensitive to light you were then). NIR light does not normally induce a PLR, because our eyes are not normally sensitive to these wavelengths of light. However, mice injected with pbUNCPs showed a dramatic PLR when exposed to NIR light, suggesting they could sense the intensity of this light!

pbUNCPs allow for detection of near-infrared (NIR) light! Above, you can see the pupils of two mice, a control mouse injected with PBS, and a mouse injected with the pbUCNPs. As you can see, when exposed to no-light, both pupils are wide, indicating that they both interpret the environment as ‘dark’. However, when exposed to NIR light (980 nm), only the mouse injected with pbUCNPs shows a pupillary light reflex (PLR), indicating that they are able to discern NIR light from darkness (an ability not possessed by control mice).  (Ma et al., 2019)

pbUNCPs allow for detection of near-infrared (NIR) light! Above, you can see the pupils of two mice, a control mouse injected with PBS, and a mouse injected with the pbUCNPs. As you can see, when exposed to no-light, both pupils are wide, indicating that they both interpret the environment as ‘dark’. However, when exposed to NIR light (980 nm), only the mouse injected with pbUCNPs shows a pupillary light reflex (PLR), indicating that they are able to discern NIR light from darkness (an ability not possessed by control mice). (Ma et al., 2019)

To further probe the question of whether the mice could see this type of light or not, they recorded the activity of neurons in the retina of mice that had been injected with pbUNCPs or PBS (control). Indeed, only retinas from mice that had been injected with pbUNCPs showed electrical responses to NIR light, indicating that these nanoparticles were able to render the retina sensitive to this normally ‘invisible’ light. Importantly, the retina from mice injected with pbUNCPs also showed normal responses to light in the visible range (535 nm), suggesting that their ability to sense NIR light did not interfere with their ability to see ‘normal’ light.

Retinas from pbUCNP-injected, but not control-injected mice respond to NIR light! The first two panels above (vertical) show how a control mouse responds to visible light (top) and NIR light (no response; 2nd from top). Reciprocally, mice with pbUNCPs respond the same to both visible and non-visible NIR light.  (Ma et al., 2019)

Retinas from pbUCNP-injected, but not control-injected mice respond to NIR light! The first two panels above (vertical) show how a control mouse responds to visible light (top) and NIR light (no response; 2nd from top). Reciprocally, mice with pbUNCPs respond the same to both visible and non-visible NIR light. (Ma et al., 2019)

This physiological evidence is great, but what about something more relevant to behavior? Can mice see well enough in NIR light to make decisions in response? To test this, the authors performed a number of behavioral tests, the outcomes of which depended on whether the mouse could discriminate NIR light from visible light. The first of these tests was a widely known and well-validated test of anxiety, the “light-dark box”. This test takes advantage of the fact that mice prefer a dark over light environment (as they are nocturnal, and do not want to be spotted by a day-active predator!). Here, the researchers shined visible (535 nm) or invisible NIR light (980 nm) into the ‘light’ chamber, and tested whether control or 'pbUCNP’-injected mice responded by running into the ‘dark’ chamber. Only mice that could see the NIR light (pbUCNP-injected) responded tot he 980 nm light by running into the dark box. The mice with just normal vision could not recognize that the 980 nm light was on, and simply explored the dark and light boxes equally. Importantly, both control and pbUCNP-injected mice avoided visible light (535 nm) when it illuminated the ‘light’ chamber, suggesting that these augmented mice could see normal light just as well as the control mice.

pbUCNP-injected mice recognize and respond to NIR light cues to elicit behavioral responses. The top two panels (C,D) show results of a light-dark box test, where mice can choose to be out in the open (in the light) or retreat into a dark box (which they naturally prefer). Control mice and those injected with pbUCNPs responded to visible light (525 nm) by retreating into the dark box, however when the light was in the NIR range (980 nm), only mice injected with pbUCNPs responded, while control mice could not discern a difference between 980 nm light and darkness. In the lower panels (E,F), mice were tested for their ‘freezing’ responses in a ‘fear conditioning’ paradigm. A 535 nm (visible) light was shown for 20s before a 2 second footshock for 6 cycles to let the mice form an associative memory (where light predicts a painful stimulus (shock)). Normal mice, after forming this memory show a ‘freezing’ or ‘immobile’ response to just the light, because they ‘remember a shock is coming’. When the researchers illuminated the mice with 535 or 980 nm light after training, control mice only froze in response to the 525 nm light, while the pbUCNP injected mice froze in response to 535 and 980 nm light!  (Ma et al., 2019).

pbUCNP-injected mice recognize and respond to NIR light cues to elicit behavioral responses. The top two panels (C,D) show results of a light-dark box test, where mice can choose to be out in the open (in the light) or retreat into a dark box (which they naturally prefer). Control mice and those injected with pbUCNPs responded to visible light (525 nm) by retreating into the dark box, however when the light was in the NIR range (980 nm), only mice injected with pbUCNPs responded, while control mice could not discern a difference between 980 nm light and darkness. In the lower panels (E,F), mice were tested for their ‘freezing’ responses in a ‘fear conditioning’ paradigm. A 535 nm (visible) light was shown for 20s before a 2 second footshock for 6 cycles to let the mice form an associative memory (where light predicts a painful stimulus (shock)). Normal mice, after forming this memory show a ‘freezing’ or ‘immobile’ response to just the light, because they ‘remember a shock is coming’. When the researchers illuminated the mice with 535 or 980 nm light after training, control mice only froze in response to the 525 nm light, while the pbUCNP injected mice froze in response to 535 and 980 nm light! (Ma et al., 2019).

To further test whether mice could really see NIR light without damage to normal vision, they used a ‘Y-shaped water maze’, where mice are put in water (which they dislike) and have to discern a triangle from a circle to escape down one arm of the ‘Y-maze’. One of the arms (e.g., the one associated with a triangle) has an elevated platform underwater that the mice naturally try to find to get out of the water. The mice are trained to know that the triangle is the right choice, and then tested at a later date to see if they remember this using shapes projected in visible (535 nm) or invisible (NIR; 980 nm) light.

Mice were tested on the ‘Y-shaped water maze’, where they had to swim to escape the water by finding a hidden platform located at the end of one of the arms of the maze. In these experiments, the triangle shape pointed the way to the hidden platform. Using various patterns of visible and NIR light, they demonstrated that only pbUCNP-injected mice could perform at levels significantly above chance (50%) when images were presented in NIR and visible light, indicating they could see not only the light, but discern discrete shapes as well.   Note : Green in the above image represents shapes shown in visible light, while red indicates they were shown in NIR light (Ma et al., 2019).

Mice were tested on the ‘Y-shaped water maze’, where they had to swim to escape the water by finding a hidden platform located at the end of one of the arms of the maze. In these experiments, the triangle shape pointed the way to the hidden platform. Using various patterns of visible and NIR light, they demonstrated that only pbUCNP-injected mice could perform at levels significantly above chance (50%) when images were presented in NIR and visible light, indicating they could see not only the light, but discern discrete shapes as well. Note: Green in the above image represents shapes shown in visible light, while red indicates they were shown in NIR light (Ma et al., 2019).

They observed that no matter where they put the triangle and circle (left or right arms of the Y-maze), or what background they used (visible, dark, or NIR light), the pbUCNP injected mice almost always picked the triangle arm of the maze, allowing them to escape. In contrast, control mice could not discern the NIR light circle from the triangle, and their performance on the task was only at chance level (50% correct). This exciting study is the first to artificially enhance vision using bio-compatible nanoparticles that self-anchor to photoreceptors in the retina (rods/cones). Although mouse vision is much different than human vision (mice primarily explore their environments using smell (olfactory) cues, rather than sight), there is no biological reason why this technology couldn’t be applied to humans as well. Whether it should be….is another question! What would you do with infrared vision? How could this change the playing field for soldiers, doctors, pilots….etc…all of whom use infrared technology for very important tasks daily? Leave a comment down below and join the discussion!!