Krzyzstof Palczewski: Well, I'm delighted to tell you about our project, which is related to two photon opthalmoscope for human retinal imaging and functional testing. This project has been directed by me and by Grazyna Palczewska and we're able to recruit several experts in different areas of imaging, primarily related to safety, like Francois Delori and Alfred Vogel, and also to other aspect that were required to build to make progress in our proposal related to metabolic two photon imaging, human instrumentation build up, and excitation of light modulation, as well as functional assay and clinical evaluation. Everybody on this list contribute tremendously to this project, but I would like to single out a few people involved in this project, namely Eric Gratton from University of California, Irvine, and Dr. Andrew Browne, who is retina specialist in our institute who was extremely helpful for human imaging. And none of this work would be possible without a tremendous help from Maciej Wojtkowski, who helped in human imaging both for light sensitivity, as well as for imaging. The two specific aims that were proposed on the beginning of this project were quite ambitious and I am delighted to report to you that both of those specific aims were fulfilled through those passing years. First specific aims was to develop mouse two photon opthalmoscope to diagnose and to perform functional images in live animals through endogenous fluorophore that are related to retinoid cycle because retinoids, particularly retinol and retinyl ester, are flourescent and we can utilize those property for diagnostic purpose of functional responses to light in live animals. The second was to develop and test two photon imaging that will have capability to quantify functional responses in humans. So both of those aims were successful, we were successful in accomplishing, and really open a new area of two photon imaging exploration for diagnosis and imaging of human retina in normal as well as disease state. The basis for both of those projects where the publication's prior publication, one in Journal of Cell Biology by Imanishi, my postdoctoral fellow at that time, who applied for the first time two photon imaging to human, to mouse eyes. What he discovered is specific structure of the retina called retinosomes that are present in RPE cells. And this really open up the possibility of images on sub cellular level. The second paper was published in PNAS in 2014 where we quantified and provide absolute proof that two photon can also cause isomerization of rhodopsin. That was a major breakthrough because it will allow us to interrogate retina with infrared light and to look for responses under condition where infrared light can combine two photons of that light can combine in terms of energy and cause isomerization of rhodopsin and cone pigments and evoke electro physiological response. So this study, again, open up possibilities of testing using infrared light of visual function. With the advantage obviously of infrared light is better transmission through even a very dense matter, and particularly with the aging eye, this technology appears to have a potential as we develop later on. And we're going to show you in a second. So this both imaging as well as a sensitivity are critical for regenerative medicine because the regeneration of certain cell types or implantation of RPE cells or photoreceptors or retina were required imaging and required a sensing whether the implanted part of the cells or tissues are sensitive to light. And I think this technology will be critical to evaluate, particularly those type of technology to rescue sight in blinding diseases. 

 Grazyna Palczewska: So eye affects our introduction with the world by collecting light and converting it to electrical signal which brain then interprets as vision. And the same window that lets the light through into the eye will also allow us to investigate processes or retina at the back of eye. And existing technologies such as SLO and OCT can provide us with the information about the structural changes to the eye, however, to assess their metabolic processes that sustain healthy vision or to assess the impact of the therapies, different methodology is needed because the existing methodologies would have to use the UV light, which is not only harmful, but only poorly transmitted through the front of the eye. We conquered that barrier by employing that quantum process may need a two photon excitation and in two photon excitation, two lower energy photons do the job of the one high energy short wavelength photon. Thus we can excite the molecules in the eye with the infrared light. After the two photon excitation, molecule can release the energy by emitting the fluorescent photons. And how it happens, the pulsing lasers deliver a very high energy during the vibrational time thus allow the two photon excitation. Moreover, by controlling the temporal properties of the pulsing laser, such as time between the pulses or pulse duration, we can improve the efficiency of two photon excitation and thus reduce the average power that is needed to image those molecules in the eye and this is extremely important for fully non invasive imaging of the processes in the eye. Furthermore, when using the pulsing laser, we can not only measure the quantity of photons that arrive at that particular image pixel but we can also measure the time delay between the laser pulse and photon arrival. Thus, we can have the grayscale images based on the photon intensity, but we also can assign the third dimension to the image based on the fluorescent decay or fluorescence lifetime. Such that each molecule can have a different color based on the fluorescence decay. And this is extremely important for fully non invasive imaging where we don't use an exogenous dyes because each molecule has the characteristic lifetime. So based on this, on this principle, we designed and assembled the system for imaging the RPE and the retina in the non invasive way. But to do it in a really quantitative manner, we also use the phasor transformation for fluorescence lifetime of the fluorescence lifetime. Thus instead of the of the decay function for every pixel, we have we converted the decay function to the point on the cartesian coordinates such that each molecule will have their own place on the cartesian coordinates and such, we use the mouse model of LCA RPE65 knockout and determine the location of the retinyl esters which are collected with retinosomes such as shown here around the border of the RPE cells. To look at the distribution of the A2E or retinal condensation product, we use the mouse models that that the show deterioration of the exposured light and we determine the position of the A2E on the Cartesian coordinates by using the 850 nanometer excitation light because the retinyl esters do not emit fluorescence in response to this excitation. That's when we use the 730 nanometers in this mice, we can see all the fluorophores in the RPE and then we know the location of the retinosomes.  We know the location of the A2E and the points that are located between the retinosoes and A2E correspond to the mixture of those fluorophores. Taking this advantage, we can also quantify how many particular components are in particular location in the RPE cell, thus we provide a non invasive biopsy of the original fluorophores and here for instance by selecting only the retinyl esters, we can quantify the surface that is covered by this retinyl esters. in the RPE65 mice and compare it to the mouse that is a model of the light induced degeneration. And we got a very good color correlation between our imaging evaluation and between this, and between HPLC measurements which you know is fully invasive as the eye does not survive that. And considering the volume here we have this measurements by using the image increase fully confirmed by HPLC. Another fluorophore that is present in the eye is the melanin and here we found the signature on the Cartesian coordinates of the melanin by imaging by fluorescences lifetime imaging of mice with melanin and in albino mouse, and here with a very characteristic location is in the choroid, because there is not that many other fluorescent molecules in the choroid. And we can use that that information to find the optimal wavelength, excitation wavelength, for imaging retinyl esters in the RPE. And we use the 730 nanometers, and found that we really have the good contrast between the retinosomes and the melanin when imaging with the shorter wavelengths below 780. When we image with 780 nanometers, the contrast between the retinosomes and the melanin disappears, and in such a way we can the image the eye in the living animal and we show that the bright spots here in the gray scale image correspond to the red dots which then correspond to the retinyl esters in the RPE. And now taking advantage of these discoveries, we have now obtained the first images in the human retina and you'll see here the two photon excitation fluorescence as well as the SLO image. And even though those are very preliminary images you already see a different level of the details that are visible when we image with two photon excitation And then here the kudos for this result. Go to Dr. Palczewski who was instrumental in obtaining these images. Two photon excitation also offers tremendous advantages to measuring the function of the retina, to measure visual sensitivity, thanks to the IR better penetration through the turbid media, but also in two photon excitation scattered total do not contribute to the visual signal. And first to validate that, we obtained the data in a mouse retina and in the primate retina using the specially constructed fixture where the retina is placed between the two plates and submerged in the solution that keeps it alive. And during this measurements, we can also image the retina so we know exactly at which location the signal is obtained. And on the right here you see the plot and as one would expect for one photon excitation, the sensitivity, the visual sensitivity drops with the increasing of the wavelengths. And we would expect it based on the old results that it would go all the way to thousand nanometer or even longer at this visual sensitivity will increase. However, when employing the two photon excitation at around 780, 750 nanometers, we start seeing stabilization of the signal or even increase when we go to 1000 nanometer. That's based on this measurements we compare that the signal from the macula, from the macula cones, contain about 85% of M-/ cones and 15% of the L-pigment. Knowing this, we constructed the system for measuring the two photon sensitivity in humans and the system contained both the visible light as well as the infrared light to measure the sensitivity and to confirm that indeed division is induced by the two photon excitation. We plotted the the visual thresholds to visible light as versus the visual threshold for infrared light or two photon light and we obtained the line with the slope of two further confirming that indeed this is the two photon vision that we are observing. And this system also allow us to take the maps of the visual sensitivity at the pre selected points on the retina. And here you see the 2D maps as well as the 3D maps with the lower sensitivity in the center of the retina. Encouraged by these results, we obtained the data in human subjects, in larger sample of human subjects, and obtained also the sensitivities dropping with the age as expected because the retinal processes in the back of the eye are not so great when you are getting older. And as expected in the retinopathies, we have also the drop of the sensitivity. And we confirm that the two photon excitation could be used to measure the visual sensitivity in diabetic retinopathy patients. Does this prove that  really does two photon excitation to measuring visual function, could be useful in the clinics and there are a couple things that make it useful. We observed that during this measurements we didn't have to do any correction for the lens density as is typically done further confirming that IR light is not being scattered in the lens. And also we confirm that patients didn't see any discomfort during the measurements with the two photon excitation. This was an extremely fruitful period, and we have published the latest paper being just published today in the key publication here is the non invasive two photon optical biopsy of retinal fluorophores which we published in 2020, then another papers in JCI Insight and TVST and in BOE. Furthermore to inspire free discussion and to promote the progress in the imaging, we also organize the Irvine 2020 Retinal Imaging Colloquim. And it was the last meeting that we had in person in February 2020. It had a variable discussion among the experts in nonlinear imaging,  retina imaging safety, intravital imaging, advancement in hardware and software, and in data analysis. 

 Krzysztof Palczewski: Two photon imaging of the eye is going to stay with us as a one of the modality, important modality, to interrogate function and structure of the retina. We're very delighted that many of the technical issues have been resolved in the last several years and now the next step would be to analyze and to test those both imaging as well as sensitivity, in cases of human diseases and also how the treatment of those blinding disorders can be monitored by those two technologies. So two photon imaging is the imaging technology that will allow us to get additional information to commonly use imaging modality like OCT that will expand the repertoire and hopefully for clinicians to utilize this technology as we go forward. Thank you very much.