We found that over the course of the day, the reflectance of nearly all cones, under long coherence illumination, oscillated sinusoidally. Moreover, we observed that the phase of these oscillations varied randomly from cone to cone. These observations were predicted by the interferometric model of cone reflectance presented in and Eq. (1)
, and thereby lend confirmation to the model. The model appears to describe accurately the reflectance of most cones under long coherence illumination. The model further predicts that no such oscillations in reflectance should occur when the cones are illuminated with short coherence light. The lack of oscillations in our short coherence trial (Trial 6) lends additional support to the model. Indeed, other investigators conducted an experiment in which the cone mosaic was imaged every hour over a 24-hour period using an incoherent visible illumination source [15
]. While that study revealed changes in cone reflectance, both rapid changes occurring over matters of minutes and slow changes occurring over matters of hours, it did not show the highly prevalent, sinusoidal reflectance oscillations we observed using the long coherence source. Neither did that study claim to observe disc renewal or its optical correlates. While the experimental protocols—regular imaging over periods of hours—were similar between that experiment and our own, the differences in illumination sources—theirs incoherent and visible, ours coherent and near-infrared—make comparisons between the findings difficult. Based on current evidence, we believe the mechanisms underlying the main changes in cone reflectance observed in the two experiments to be disjoint. In terms of Eq. (1)
, changes in the cone's brightness due to temporally incoherent illumination are described by Io
, whereas temporally coherent effects are described by 2 | Ψ1
) . We determined that the main changes we observed in cone reflectance, viz. sinusoidal oscillations, were an effect of temporally coherent illumination. We did not observe the temporally incoherent effects observed by Pallikaris et al. There are many potential reasons for this: the incoherent cone effects they observed may require the visible stimulation of cones present in their experiment but not ours; alternatively, these effects were present but undetectable because they were either masked by the high amplitude coherent effects or the relatively low cone SNR imposed by near-infrared illumination [28
Using the outer segment as a biological interferometer, we were able to track minute changes in its length, significantly smaller than the wavelength of the illumination source. We found that the rate of renewal can be determined within five hours and that renewal can be continually tracked over 24 hours while monitoring small but significant changes in its rate over the course of the day.
While it has not previously been possible to measure OS renewal rate in living animals, let alone humans, numerous ex vivo studies have been conducted to study these rates in mammalian rods. Daily rod renewal rates have been reported to be 1.8 to 2.2 μm /day in mice [6
], 1.8 to 2 μm /day in dogs [31
], and 2.6 to 2.8 μm /day in rhesus monkeys [33
]. In mammalian cones, electron microscopy has been employed to measure the size of phagosomes in the RPE cells underlying the cone OS, found to be 1.6 μm /day in the squirrel [11
], 1.15 um/day in the domestic cat [34
], and 1 to 3.23 μm /day in the Rhesus monkey [35
The rates of renewal we report here lie squarely within the range of renewal rates reported in the literature and agree with the estimates of daily disc shedding in cones, suggesting that we have developed an accurate method for measuring disc renewal in the living human eye. This is the first observation of renewal in a living eye and the first direct observation of renewal in cones. While the measurement period is currently long (5 hours), improvements are possible to significantly shorten it, which will enhance the technique's practicality for measuring larger numbers of subjects.
Our technique could be used to measure normal rates of outer segment renewal in a population study, and given such normative data, be extended to investigate the relationship between renewal and disease. Defective phagocytosis of outer segments has been shown to play a role in retinitis pigmentosa (RP) [37
]. Disruptions of phagocytosis and associated signaling between RPE and photoreceptor has been shown to play a role in age-related macular degeneration (AMD) [38
]. Measuring rates of renewal in AMD and RP patients’ retinas could help determine the role of disc renewal in these diseases, and may be useful in early detection and monitoring of progression in these leading causes of blindness. Moreover, the method could be employed to investigate the cyclic properties of renewal and shedding in human cones and the governing roles of circadian and light cues, none of which has yet been characterized. Recent years have witnessed considerable interest in optical measurements of photoreceptor function; see, for example [12
]. While the dependence of electroretinogram (ERG) signals on daily renewal has been measured [48
], it would be interesting to use the current system to explore the relationship between renewal and stimulus-evoked optical changes, at the level of single photoreceptors.
The best existing method for depth imaging of the living retina is ultrahigh-resolution optical coherence tomography (UHR-OCT), whose axial resolution has been reported to be 3 m in retinal tissue [49
]. Combined with AO, UHR-OCT can measure outer segment lengths of individual cones [50
]. However, the axial resolution of UHR-OCT is likely insufficient to detect daily OS renewal, let alone that which occurs over matters of hours or daily variations in rate. Spectral-domain phase microscopy has demonstrated sub-nanometer resolution in in vitro
], but has not been demonstrated in vivo
, likely because of the substantial phase noise introduced by eye motion even when the subject's head is well stabilized. Our instrument may be thought of as a phase-sensitive common-path interferometer that uses the two opposing sides of the structure of interest (OS) as sample and reference surfaces. As such, the instrument is sensitive only to those phase changes that occur between the two surfaces. While we have targeted disc renewal, our method can be readily applied to other physiological processes that exhibit minute length or refractive index changes in the OS, as for example the processes of disc shedding and phototransduction.