We demonstrated the performance of our SLO/OCT instrument to image and observe in vivo the three dimensional structure of human cone photoreceptors over extended periods of time. This observation is only possible because the registration between the recorded 3D data sets is better than the extension of a cone (transverse) and better than the coherence length of the light source (in depth). We investigated changes in the backscattered intensity of individual cones and at different retinal layers for two different time periods: 7 hours and 96 hours, respectively. Previous studies investigated intensity fluctuations using AO-FC [19
] which showed similar results as our SLO and depth integrated OCT images. However, our study showed that intensity fluctuations within individual retinal layers (IS/OS, OS, ETPR, RPE) are much more pronounced than in the depth integrated image (that was investigated using AO-FC). Interestingly, we found that the intensity fluctuations within a cone at the IS/OS and the ETPR are rather independent from each other (c.f. ). Therefore we can now directly confirm the suggestion made in Ref [19
] that a change in the pointing direction of a cone cannot be the origin of these intensity fluctuations because in that case the intensity at both layers would be affected similarly. In Ref [19
] it is speculated that the intensity changes mainly origin from the reflection at the ETPR. However, our results show that in both layers intensity fluctuations can be observed. The origin of these intensity fluctuations remains therefore unclear. Nevertheless we think that the intensity fluctuations at the IS/OS could be caused by the permanent generation of new discs at the IS/OS layer which may result in a change of the refractive index difference at this interface. Depending on the phase within the disc generation process high or low backscattering signal is observed. Noticeable is that the BRS (located within the OS) do not change their intensity within the 7 hours period. This strengthens the above mentioned hypothesis that the intensity fluctuations are caused only by changes that affect the interface between inner and outer segments of cone photoreceptors. Similar, the intensity fluctuations observed at the ETPR could be explained by different status of the disc shedding process. However, within the 7 hours period no change in the OS length is observed (in contrast to the 96 hours measurement) therefore it is most likely that during this period discs from the majority of the cones were not shed. Although there is no data on human cone disc shedding, results from animal studies show that in some species the shedding process is determined by a circadian rhythm [25
] with a peak of disc shedding shortly after light onset [14
], a period that has not been investigated in our study. Note that the OS length changes observed in the 96 hour experiment are very small (up to a few µm) which is in agreement with the observed size of phagosomes (that would correspond to the expected length changes) in animal studies [13
Still unclear is the origin of bright reflections at the transverse location of individual cones within the RPE (c.f. movie in ) that are changing with time. Although we can, at the current state of investigation, only speculate on the origin, it might be plausible that light is backscattered at migrating macrophages (probably containing phagosomes) or pigments. Depending on the position of these particles in respect to the overlying cone, light can be back-coupled into the photoreceptor which finally leads to our observation. Interestingly, the cones under which theses reflections can be observed are randomly changing. Although at the depth location of the RPE (depth integrated over 12µm around the peak of the RPE) each en-face image shows a random pattern (c.f. ) the full cone mosaic can be observe when all images recorded over the measurement period of 7 hours are averaged (c.f.
). This strengthens the assumption that light originating from this layer is back-coupled through the photoreceptors (at least at some points in time).
Fig. 17 En-face image of the RPE, averaged over the measurement period of 7 hours (depth integrated 12µm around the peak of the RPE position) OCT images. (Image extension: ~0.94°x0.7°, retinal eccentricity: ~4° nasal from the fovea) (more ...)
As shown in the previously reported BRS [16
] are changing their position within the OS when imaged over an extended period of time (96h). Although we hypothesize that the BRS originate from defects or cracks within the packing of the OS discs, a proof of this hypothesis is very challenging. Animal studies including histology or in vitro multi-photon imaging could probably be of help here, although a perfect registration between the different methods might be very difficult.
The measured motion speed of the BRS corresponds well with the cone renewal speed known from animal studies [23
] and from AO-FC measurements [15
]. Note that both in vivo methods do require measurement series over several time points. While our method measures the renewal speed over an extended time (5 measurements in 96h), the AO-FC method requires several measurements with shorter time intervals (every 15 minutes up to a total time of 5 hours). Therefore two (probably?) different renewal speeds are measured. With our method a mean renewal speed (averaged over 4-5days) is measured while with the AO-FC method a renewal speed within 5 hours is measured.
Finally we want to point out that the results presented in this paper are based on 2 volunteers. Certainly more subjects have to be imaged in order to strengthen the presented hypotheses.