"Mouse fuss" in infrared light: the introduction of nanoparticles in the subretinal region of the eye of a mouse

The world around us is full of information in various forms of its manifestation. It does not matter where you are: indoors or outdoors, in the city or among the fields, in the tropics or in the snow-covered tundra. Always and everywhere your brain gets some information. By itself, this body, if exaggerated, is useless in the matter of informing you about the environment. To help him we have our senses (eyes, ears, tongue, nose and skin). However, far from all the information we are able to perceive, in particular, infrared radiation, which is not visible to our eyes. At least it was before. Today we will get acquainted with the study in which an ordinary mouse was endowed with the ability to see infrared radiation of the near field through nanotechnology. How did scientists achieve this, how did the mouse feel after the “improvement” and what are the prospects for this discovery for humans? We will look for answers to these and other questions in the report of the research group. Go.

The basis of the study

Human vision is not the best, but not the worst, among the inhabitants of the planet Earth. It would be more correct to say that it has certain limitations. We are able to perceive "visible light", that is, radiation in the range from 400 to 700 nm. But here the infrared radiation of the near field (hereinafter NIR) is above the upper limit of 700 nm.

If you dig a little deeper, the problem lies in the structure of the mammalian eye, that is, you and I. In the eye there are photoreceptors - photosensitive sensory neurons of the retina. Inside the cells there are opsin * and rhodopsin * , which play one of the most important roles in the perception of light, that is, in vision.

Opsins * - receptors associated with G-proteins, located in photosensitive cells of the retina.

Rhodopsin * - protein, the main visual pigment contained in the rods of the retina.

This whole group of receptors is aimed at capturing light, that is, photons. But with NIR radiation, everything is much more complicated. In the case of NIR, photons have lower energy. Consequently, opsins must have a lower energy barrier in order to perceive such photons. Otherwise, there is only a strong thermal noise. In other words, mammalian photoreceptors are simply physically incapable of “capturing” light above 700 nm, in particular, NIR radiation.
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But we all know that true scientists cannot be stopped by any physiological limitations. You can solve the problem with photoreceptors by giving them assistants in the form of nanoparticles that will perform those functions that are not available to natural cells (organs, systems, etc.). This scientists did in their study. They developed special nanoparticles with a built-in autonomous source of light radiation, which can extend the range of the mammalian visual spectrum (in this case, mice). Scientists explain that pbUCNP nanoparticles (retinal photoreceptor binding upconversion nanoparticles) are kind of energy converters. They convert the infrared radiation of the near field to shorter wavelength visible radiation.

The mouse eyes were “modified” by subretinal injection (under the retina) of nanoparticles diluted in sodium phosphate buffer. The use of electroretinography * and data on visual evoked potentials * from the visual cortex of the mouse helped to establish the fact that the retina and visual cortex were quite active when exposed to external stimulus in the form of infrared radiation. Simply put, a mouse with embedded pbUCNP nanoparticles reacted to NIR radiation.

The evoked potential * is the electrical response of organs to an external irritant.

Electroretinography * - the study of the state of the retina through the registration of biopotentials that occur during light stimulation.

Were also conducted behavioral tests, which also confirmed the above statement. In addition, scientists tested the biocompatibility of the nanoparticles and the mouse body, which showed only minor side effects. We will talk about the results of tests, tests and data analysis a little later, but for now we should get acquainted with what these amazing nanoparticles consist of.

PbUCNP structure

The main task of nanoparticles was the conversion of infrared light (more than 700 nm) into visible. Considering that the human eye is most sensitive to visible light with a wavelength of 550 nm, so-called conversion (or up-conversion) UCNP nanoparticles ( 1A and 1B ) were created.

If we talk about photons, then up-conversion is the process of converting several photons with lower energy, that is, with a long wavelength, into one photon with higher energy, i.e. with a short wavelength. Namely, this is necessary, given the properties of the photoreceptors of the mammalian eyes.


Image number 1

These nanoparticles exhibited a peak of the excitation spectrum at 980 nm and a peak of radiation at 535 nm with a light exposure of 980 nm ( 1C and 1D ).

To obtain water-soluble nanoparticles, scientists applied concanavalin A protein (ConA) to the surface of paaUCNP particles, that is, coated with polyacrylic acid ( 1E ). The use of ConA is justified by the fact that this protein binds perfectly to sugar residues and derivatives of the outer photoreceptor segment, resulting in the formation of glycosidic bonds * .

The glycosidic bond * is a covalent bond between a sugar molecule and another molecule.

It was necessary to check the success of the implementation of ConA on the surface of the UCNP. To do this, scientists have added b-cyclodextrin to a solution with pbUCNP, which possesses the same glucosyl unit as the external segment of the photoreceptor. As a result, there was an aggregation (combination) of ConAb-cyclodextrin (TEM image in image 1G ).

Therefore, similar observations confirm that pbUCNP will be able to successfully attach to the surface of the mouse photoreceptors.

The TEM image of 1H shows that pbUCNP retains its monodispersity when b-cyclodextrin is added, and this is because ConA is absent in this combination. The introduction of such pbUCNP into the subretinal region of the mouse eye ( 1F ) resulted in the nanoparticles binding to each other, forming a close relationship with both the inner and outer portions of cones and rods ( 1J , 1K and 1L ). In this way, a layer of nanoparticles with a characteristic up-conversion spectrum was formed (image on the left at 1I ).

If paaUCNP particles were introduced into the subretinal region of the mouse's eye, they formed very weak links, so they could be easily removed from the photoreceptors (the picture on the right of 1I ).

The biocompatibility check did not show any major abnormalities. To test the mouse, a simple solution of sodium phosphate buffer (without nanoparticles) was also injected into the subretinal region of the eye of the mouse - no difference was found. Those side effects that were discovered were associated with the most subretinal injection and completely disappeared within 2 weeks after the procedure.

Checking the integrity of the retina and a healthy amount of photoreceptors showed that even with the introduction of 50 mg of pbUCNP in each eye, no negative changes were observed. That is, the structure of the retinal layers does not degrade (this is seen in images 2A and 2B ).


Image number 2

The scientists also decided to check for the presence of inflammatory processes in the retina of the rat using the microglia marker Iba1. The analysis showed a slight inflammation of the retina at the 1st, 2nd, 4th and 10th week after injection of pbUCNP ( 2C and 2E ).

In addition, apoptosis (disintegration) of retinal cells was tested after injection by labeling with terminal deoxynucleotidyl transferase (TUNEL). TUNEL signals were detected only 3 days after the injection of both pbUCNP and pure sodium phosphate buffer ( 2D ). On the 1st, 2nd, 4th and 10th week after the injection, no signs of TUNEL were detected ( 2E ).

Summarizing the results of the above analyzes, the scientists came to the obvious conclusion - pbUCNP does not harm the body of the test (mouse), except for certain side effects caused solely by the procedure of injection into the subretinal region of the eye.

Now that we know what pbUCNP is and how it affects the health of experimental mice, we can proceed to review the results of practical tests of the performance of pbUCNP nanoparticles.

Research results

Image number 3

To check the reaction of photoreceptors to infrared radiation, samples of rods were taken from the retina of the mouse with nanoparticle injection and the mouse without it ( 3A ).

The sticks of mice that were injected with pbUCNP showed a normal photocurrent caused by visible light at 535 nm, in contrast to mice without an injection ( 3B without injection and 3D with an injection).

When exposed to a flash of light at 980 nm, a response was obtained only from the sticks of mice with pbUCNP ( 3E ); the sticks of ordinary mice did not react at all ( 3C ). It is also worth noting the strong similarity of the amplitude and kinetics of the photocurrent in the rods of mice with pbUCNP when exposed to 535 nm and 980 nm light ( 3F , 3G and 3H ). And the ratio of time and peaks suggests that there was no delay in the activation of rods when exposed to infrared radiation. It was also found that the sticks (after injection) quickly adapt to infrared light, as is usually the case with visible light.

The electroretinogram (ERG) of mice with and without injection also confirmed the fact that the response to infrared radiation at 980 nm. The results of the ERG of pbUCNP mice strongly resembled the results when exposed to visible light (535 nm). The control group of mice (without nanoparticles) had no reactions.

In addition, scientists conducted a test with mice that did not have sticks. This test showed the activation of cones by radiation at 980 nm by exposing them to pbUCNP nanoparticles.

After conducting laboratory tests, scientists have moved to the test in practice, so to speak. That is, they decided to personally observe the behavior of mice with and without injection in special conditions.


Image number 4

For a practical experiment, two boxes were used: dark and illuminated by radiation in the visible and infrared range ( 4C and 4D ). The second variant of the experiment is based on the relationship of the light stimulus and the feeling of fear caused by it ( 4E and 4F ). And now more detailed about each of the experiments.

In the first test with a dark and illuminated by visible light box of the mouse, of course, preferred to be in the dark. Visible light was replaced with LED with a wavelength of 980 nm. In this embodiment, mice with nanoparticle injection continued to choose a dark box, rather than an illuminated one, following their innate instincts, so to speak. But the control group of mice (without injection) did not see any difference between the two boxes (dark and with 980 nm light), since they literally did not perceive infrared radiation.

The second experiment was related to the study of even more thoughtful actions of mice. At the preparation stage, mice from both groups were taught that, following a 20-second emission of 535 nm light (visible), a two-second minor electric shock ( 4E ) would follow. In response to such an irritant, a completely natural response in mice should follow — numbness * .

Reaction stupor * - in some species of animals, which are usually prey, there is a protective mechanism (the last chance, so to speak). If the predator has already attacked them, they can "pretend to be dead" (stupor), thereby confusing the attacker and, having caught the right moment, escape.


How the mice react in case of danger (numb, hide or aggressively shaking the tail).

During the actual testing phase, light radiation was applied at both 535 nm and 980 nm. As a result, mice injected with pbUCNP showed a stupor reaction in both types of light exposure, as they expected electric shock. But mice without nanoparticles did not react to infrared radiation. And this suggests that they did not perceive it during the preparation and, therefore, could not link the radiation invisible to them with the shock of the current. The control group of mice had a response only to light in the visible range. Image 4F shows a comparison of the results of this test in the control group of mice and in mice with injection.

These practical tests confirmed the fact that mice with pbUCNP perceive infrared radiation, but can they really see in such a range, in the truest sense of the word? In order to get an answer to this question, scientists conducted another test - measuring VEP, i.e. visual evoked potentials ( 5A ). To do this, electrodes were installed in six areas of the mouse visual cortex (No. 1, 2, 3, and 5 in the monocular areas and No. 6 in the binocular areas).


Image number 5

When visible light affected the eyes (535 nm), a reaction was observed in all areas of the visual cortex in all mice (with and without nanoparticles), which is quite expected ( 5B and 5D ). But with the light at 980 nm, the mice were divided into two groups, as in previous tests. In mice with injection, VEP was found in the binocular areas of the visual cortex ( 5C and 5E ). No VEP was identified in the control group. It should be noted that the manifestation of VEP in the binocular areas is consistent with the injection site (the temporal side).

At this "mouse fuss" is not over. The next test again was a more practical test with a water labyrinth in the shape of the letter “Y” ( 6A ), according to which mice with pbUCNP had to be oriented by infrared radiation.


Image number 6

During the preparation, the mice were trained to find a hidden platform that was connected to one of the two maze routes. In total, scientists made 5 variants of the test with different visual stimuli and light emission.

In the first version there were light grids ( 6B ), the position of the lanes on which indicated the direction of movement. Mice with nanoparticles successfully learned to distinguish the orientation of the bands (vertical or horizontal) and saw them perfectly when exposed to light radiation at 980 nm. The control group chose the platforms in random order, that is, could not distinguish them from each other due to the inability to see in the infrared spectrum ( 6C ). In the test where visible light was used (as during training) both groups of mice successfully coped with the task.

Measurement of the wave number (spatial frequency) showed that in mice with injection, this is 0.31 ± 0.04 in visible light. In mice from the control group, this indicator is equal to 0.35 ± 0.02, that is, there are no particular differences between the two groups of subjects. Therefore, the introduction of nanoparticles into the retina did not affect how mice perceive visible light. In the case of infrared radiation in mice with pbUCNP, the above indicator was 0.14 ± 0.06. Scientists associate such a decrease in spatial frequency with isotropic radiation and the scattering of visible light from nanoparticles excited by infrared light ( 6D ).

In the second version of the test, the scientists decided to check whether the perception of infrared radiation interferes with the photon radiation in the visible range. Two plates with visible (535 nm) and infrared (980 nm) radiation LED matrices that are perpendicular to each other were made. When all the LEDs were turned off, both plates looked identical against the background of visible light ( 6E ).

During preparation, the test chamber turned on the lights (visible light, 196 lux) and only 980 nm LEDs. During the actual test, only injection mice were able to successfully recognize the plates ( 6F ). This suggests that their perception of infrared radiation has not deteriorated due to the background radiation of visible light. In the case when only 535 nm LEDs were turned on, both groups of mice showed good results, as expected.

The next test was to recognize triangles and circles ( 6G ). PbUCNP mice successfully distinguished shapes in visible and infrared light when the test chamber was unlit, that is, in the dark ( 6H ). The control group could only detect shapes from visible light.

After that, one more variable was added to the task - the background light, but not visible, as before, but infrared. Mice with pbUCNP still distinguished infrared / visible light patterns with background infrared radiation.

In the final test, the scientists decided to find out whether mice with an injection can see figures in the infrared and visible range at the same time. In this test, there was a water maze with platforms on which a circle and a triangle were simultaneously depicted. During the preparation, only triangles in visible light were active. But during testing there were triangles and circles (980 nm) in a random sequence (6I). As expected, mice with pbUCNP perfectly distinguished the figures (6J). Checking the results of this test in both groups of test mice confirmed that mice with an injection did not choose this or that platform randomly, in contrast to the control group. Thus, it is possible to fly the conclusion that the injection of pbUCNP allows mice to see objects both in the infrared and in the visible range at the same time.

For more detailed acquaintance with the nuances of the study I strongly recommend to look into the report of scientists and additional materials to it.

Epilogue

Such a study is excellent evidence that nanotechnology can be applied in very different directions. Of course, it’s too early to say that their possibilities are endless, but every day we get more and more new ways of applying nanotechnology. In this particular case, the use of nanoparticles in order to donate mice with infrared vision is not only an amusing experiment, but also a confirmation of the unique capabilities of the implemented improvements in biological systems. Scientists themselves are not yet ready to make loud statements about the application of their development in medicine or in any other areas, but they will continue their research in order to improve the above-described nanoparticles and, possibly, give them new properties.

Be that as it may, the widespread use of augmentations by a person to improve and change his body will not happen soon due to not only the imperfections of technology, but also a multitude of ethical questions that many public figures have already asked. Is it possible to allow a person to change his body? Where is the limit of allowable augmentations? How will this affect social class stratification in society? Does this create new conflicts in an already conflict world? The list of such questions can be continued, but no one has yet given clear answers to them (the games of the Deus Ex series do not count). Perhaps the principle of "time will tell" fits here as well as possible.


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