 Good morning, happy Wednesday. We're gonna get started. Our first presentation this morning is Dr. Lydia Sauer. She's gonna give us a presentation on clinical advances in fluorescence, lifetime imaging, and ophthalmology. Let's welcome her. Thank you very much. I'm really excited to be here. And thank you for the introduction. Does it work now? Perfect. These are my financial disclosures. And since it's very early in the morning, I would like to start with this picture that I took a couple years ago. And when we look at it, we see what's going on. We see it's a gecko that's just hanging out there enjoying its day. But do we really see all the information that we could possibly see? Or does this give us a much more detailed view of the picture? In the same way, we are very used to looking at these images in ophthalmology. This is a typical fundus autofluorescence intensity image. But when we look at flio images, suddenly we get a much more detailed view. And I'm gonna go a little bit further. This is a healthy eye. But I would like to look at some diseases. So here we see an eye with drusen due to macular degeneration. And we are very familiar with these pictures. But when we look at the flio, we suddenly get a completely different view of this picture. Or I have one more example. This eye almost looks healthy when we look at it. It's very difficult to delineate any pathology in this eye. But when we then take the flio and look at it, suddenly we clearly see something that we didn't see before. So this is what my talk is going to be about. It's about flio. Flio is a novel imaging technology that was developed in Jena and then produced by Heidelberg Engineering. The devices look very similar to the regular Spectralis OCT. But there's a lot more going on in the device. And it actually provides us a very high contrast picture of the back of the eye. And we can really delineate different information about fluorophores within their environment. And when we think of fluorescence, I'm just going to go back to the basics for a very brief moment. We can think of three dimensions in fluorescence. The first would be the spectral dimension. So when we think of the chemical view of things, the emission spectrum and the absorption spectrum and the emission spectrum are different in the fact that the emission spectrum is shifted towards longer wavelengths, which we see right here. When we come from an ophthalmological background, we always think of the brightness dimension first because that's what we're most used to, to the fundus outer fluorescence intensity images that just show us how bright do the fluorophores come back? And what I would like to focus on today is really the third dimension, the lifetime dimension, which essentially tells us how long do the fluorophores glow? So how long does it take for the fluorescence to fade away? And for that, there's a very nice little presentation that Chantal, a good friend of mine made, and I would like to show this to just highlight it a little bit more. So when we think of fluorescence, we count the fluorophores that are coming back. Doesn't really matter when they're coming back, some come back early, some later, but really what we're looking at is how bright are they? And this is our typical outer fluorescence image. When we look at flio, we give these times a color. So the fluorophores that come back first, we put in red color and say this is a short lifetime because they're coming back much faster. And then as time passes, we shift to longer lifetimes and the lifetimes turn to blue color. And this essentially transforms the outer fluorescence intensity image to the lifetime image. And we think that we get more information because in intensity imaging, it's dark at the phobia and dark at the optic nerve, whereas in flio images, we can clearly see differences between these areas. How it works is we have a 473-nanometer laser. It's a pulse laser that we send into the eye together with an IR laser for eye tracking. And then we collect the fluorescence that is coming back, but we actually split it up according to the wavelength of the fluorophores into two spectral channels. So a short spectral channel from 498 to 560 nanometers and then a long spectral channel from 560 to 720 nanometers. And this essentially gives us with one two-minute measurement these four images. So two outer fluorescence intensity and two outer fluorescence lifetime images. And when we think of healthy eyes, we always see the same pattern in these eyes. We see that they're long lifetimes at the optic nerve, which are depicted in blue and I actually added those curves to it. What you can see is the curve is much less steep. So it takes much longer until the fluorescence comes back. The intermediate lifetimes that are depicted kind of an orange-ish-greenish color that can be found all across the fundus are caused by the retina pigment epithelium and the lipofustin. And the short lifetimes in the center of the phobia are caused by macular pigment and the decay of the fluorescence is really steep. So going from this healthy eye and looking at the variety of different diseases that we can look at in ophthalmology, we clearly see that there are a lot of patterns that we can map out with flio that really look different from another. And I would like to go through a few of these diseases today just to point out really the highlights of what we think is where flio is really beneficial. And the first thing that I'm gonna start with is the whole question about macular pigment and how can we see that it's really macular pigment that we're imaging want to start with albinism because patients with albinism do not have any macular pigment and they also do not have a formal depression. And when we think of, or when we look at the flio images, we see that the short lifetimes in the center are just missing. So we cannot see them because they're absent. And we did some more studies looking at macular pigment, for example, in macular holes, in mactail, in other diseases, and we really were able to find out that the macular pigment is really causing these short lifetimes in the center. And the first disease that, or the second disease that I'm going to hit on is macular degeneration. It's a very common disease that we see in retina clinic all the time. And as I showed in the beginning, we really see this ring of blue that is a shift towards long lifetimes around the phobia. And we do not see any indication of this in the outer fluorescence intensity image. We see this ring in all patients with AMD, which is pretty striking. And we also did some statistics on it. We kind of produced a grid onto the back of the eye and looked at the area of that pattern, which is basically this ring right here. And we compared the lifetimes, not just between AMD eyes and healthy eyes, but also between AMD eyes of different stages, because we think that pattern actually progresses as the disease progresses. And we were able to find a significant difference for this pattern, whereas there was no significant difference in the phobia. And that really indicates that something must be going on in this area right here where we see that pattern. And it could be a deposition of something, for example, some bisrhodenoids or sub-RP deposits, but it could also be a metabolic change that we see. Because when we actually looked at our healthy group, and this is kind of interesting, that about 30% of our healthy subjects that were age-matched already showed a trace of that pattern. And we think that these patients were, a lot of them were of risk to develop macular degeneration. And we think that with FLIO, we might be able to see the first changes of this in the eye even years before macular degeneration is developing. So we're trying to do more analysis on this to really understand it, and especially follow-up measurements. And we also looked at neurovascular AMD. We found the same pattern, but what we saw is that sometimes in neurovascular AMD, the pattern is a little disturbed, especially when we have pigment epithelial detachments that are quite large. And we did a study just kind of looking at different PEDs. And we found that hemorrhagic PEDs at the shortest lifetimes, indicating that it might be blood that we see that has short lifetimes. And for that to kind of prove it, or kind of give another argument for it, we had this very interesting patient that presented with a fibrovascular AMD and came up, came to the office two weeks later with a new hemorrhagic PED. And what is really striking is how the wet, oops, sorry about that, how the wet in the center really increases. And we actually took his blood and did an ex vivo measurement of the blood, which you can see right here. And if we put the colors exactly to the flio image in vivo, you see that the blood is just completely wet. But if we increase the contrast a little bit, we can see that especially the erythrocyte sediments have the shortest lifetimes, which just helps us understand these pictures a bit more. And another disease that we are really excited about is macular telangulctasia type two, or in short, mactel. It's an inherited retinal disease that was initially believed to be pretty rare, but we keep finding more and more patients with this disease. And it was initially believed that it kind of starts between 40 and 60, but we keep finding younger patients that also have mactel. And it's fairly difficult to diagnose it with some imaging modalities, just by looking at the funders, because it can present similar to a lot of other diseases, especially AMD. So many patients have been misdiagnosed with AMD. And it's really valuable to have a tool that really lines out that this is mactel. So in patients with mactel, we see this prolongation of lifetimes at the temporal site of the fovea in all of the patients. It can sometimes present also as a wing. And when we compare different imaging modalities, we can really see that we can see mactel much better in flio than in a lot of the other ones, especially in early stages. The only other modality that shows mactel quite as well would be fluorescein angiography, but that again is invasive, whereas flio is a non-invasive two-minute picture, basically. So it really has an advantage here. And I would like to go a little bit further and present a very interesting family, because here in this family, the coven was diagnosed with mactel at age 21. And he is severely affected with the visual acuity of 2100. And what was interesting in this family is that they were diagnosed with chakumari tooth. And his father came in in his 50s, I believe, and he had early onset macular degeneration, is what it said. He also did not have any arms because he had burned them off in a barbecue accident. So it was all kind of very interesting to look at that family. And by looking at it a little bit further, we realized that also the coven has some peripheral neuropathy, which is the reason why the father lost his arms. So we investigated it a bit further and we were able to find a gene, HSAN is the gene mutation, but it's a neurological disease that causes peripheral neuropathy, but it's connected to the eye disease, to mactel. So we recruited a couple more patients and the patients with that mutation also have mactel. And going back to that family, the proven had two sisters. The middle sister was 26, the oldest was 28, and the middle sister actually had the mutation, but the oldest sister did not. Both of their clinical images looked completely normal, but the affected sister actually showed only inflio changes in the lifetimes, which you can see here in the wing, that is the way we can already see a trace of that mactel pattern, which could help us treat her much earlier. And we're really excited about this case, and this was recently published in the New England Journal of Medicine, as well as in Retina. So if you want to learn more about that, we're really happy about that. And we think that this really helps treating these, or approaching mactel a different way by understanding the causes behind it. So we go further and look at other family members trying to really map out which of the individuals could have early mactel changes, which wouldn't, and we found that very early mactel can present kind of in the superior area, and that some of the individuals, we just called flio positive, and others we called flio negative, and we will follow up on these to really see if we can detect mactel earlier. Here's another example of a family, the father had mactel, and two of the sons had flio positive findings, the other two had normal findings. So we'll follow up on these and see. Now of course we're wondering what is causing these long lifetimes. In HSN, we think it's a sphingolipid disturbance in the sphingolipid metabolism, and we think there could be toxic dysoxys sphingolipids that accumulate in the eye, and when we look at other patients, we see these crystals in the eye, and they really map out to the areas of long lifetimes. We try to look at progression, these are studies that are just ongoing, but really going back to the accumulation of things in the eye, this is another patient that presented in Dr. Hartnett's clinic, and it's a 14-year-old male that had a cherry red spot, and complete normal vision, it was a normal health examination where they found a cherry red spot, referred the patient to Dr. Hartnett, and we clearly see in flio that there are very much prolonged lifetimes, especially in the short spectral channel, which is kind of similar to what we see in mactel, and looking at his family a little bit further, we actually saw that his father has mactel. So is this a coincidence that a rare disease such as mactel correlates with this other disease, which we later found out was cyanlidosis? We're still investigating that. All right, I have a few more very short diseases that I'm gonna hit on. The first one is Stagard's disease. What we see in this disease is it's a retinal dystrophy that kind of presents with flecks, and I want to draw your attention really to this spot. If we look at the baseline imaging, it's red color, so short lifetimes. If we look at the follow-up, it shifts to blue. When we look at the outer fluorescence image, there's no trace of that flag in the baseline measurement, but it is there in the follow-up. So we can see how the structure progresses, but then also the patient reported a new scotoma right at this spot, and we can already see changes in the flio much earlier, so we think that with flio we can not only predict progression of structural things, but also of function. We also look at retinitis pigmentosa, investigate the wings that we see in outer fluorescence. We see them very pronounced in flio and some patients, and we're currently doing follow-up measurements. Carl Anderson has been really invested in that, and we think that at least from the first analysis, we think that the patients with the strong wings in flio are more likely to progress, but these studies are still ongoing. We also looked at plaquenol toxicity, plaquenol is an anti-inflammatory drug that is used for lupus and rheumatoid arthritis, and the problem is that it can cause irreversible eye problems, and we really try to find out, not only if we can see these damages, but also if we can see them early. So for example, this patient presented in the clinic, she was on plaquenol for 20 years and had a complete normal imaging, but had some reduced contrast sensitivity, some visual disturbances. She was taken off the drug, and the only thing where we really saw a difference was really in the flio, and we see that in some of the other patients that are on the drug as well. And skip this one. We look at more diseases. We have a lot more ongoing studies right now, and there's a lot more literature, if you would like to read up on that. But in the end, it's a new dimension of autofluorescence imaging. It's non-invasive and fast, and we basically see these really stable patterns in healthy eyes that change in early stages of diseases, and that then show us characteristic patterns in eyes with diseases. We think it's helpful to differentiate between the individual diseases. We think it's a suitable measurement method to follow up, especially on therapeutic or monitoring, and it's a possible tool for early diagnosis. And in the end, we're just beginning to fully understand the potential of this method. With that, I would like to thank you for your attention and thank all the collaborators, especially Dr. Weinstein, my mentor here, as well as Dr. Hammer from Jena and everybody else who's contributed to this. Thank you very much.