Functional optical coherence tomography enables in vivo optoretinography of photoreceptor dysfunction due to retinal degeneration.

Stimulus-evoked intrinsic optical signal (IOS), which occurs almost immediately after the onset of retinal stimulus has been observed in retinal photoreceptors, promises to be a unique biomarker for objective optoretinography (ORG) of photoreceptor function. We report here the first-time in vivo ORG detection of photoreceptor dysfunction due to retinal degeneration. A custom-designed optical coherence tomography (OCT) was employed for longitudinal ORG monitoring of photoreceptor-IOS distortions in retinal degeneration mice. Depth-resolved OCT analysis confirmed the outer segment (OS) as the physical source of the photoreceptor-IOS. Comparative ERG measurement verified the phototransduction activation as the physiological correlator of the photoreceptor-IOS. Histological examination revealed disorganized OS discs, i.e. the pathological origin of the photoreceptor-IOS distortion.


Introduction
As the center of phototransduction, retinal photoreceptors are responsible for capturing and converting photon energy to bioelectric signals for following visual information processing in the retina. Photoreceptor dysfunctions have been frequently reported in age-related macular degeneration (AMD) [1][2][3], retinitis pigmentosa (RP) [4,5], and diabetic retinopathy (DR) [6,7]. Particularly, retinal photoreceptors are known as the primary target of early AMD and RP. AMD is a multifactorial cause of photoreceptor degeneration and accounts for more than one-half of all blindness in industrialized countries [8]. The prevalence of AMD rises exponentially with age; mild to moderate AMD occurs in 30% of individuals over 75 years old [9,10]. RP refers to a group of inherited forms of photoreceptor degeneration and genetically heterogeneous, which can cause blindness even in the early age of 30 [11,12]. It is well known that early disease detection and objective assessment of therapeutic outcomes are essential to prevent vision losses and blindness.
Traditional fundus photography can provide valuable morphological information for eye disease detection and management. However, the fundus examination itself is insufficient for early detection of photoreceptor dysfunction [13]. In principle, physiological abnormality precedes detectable morphological changes, such as retinal cell loss and corresponding thickness change. Electrophysiological methods, such as focal electroretinography (ERG) [14][15][16][17][18] and multifocal ERG [19,20], allow objective assessment of retinal function. However, it is difficult to correlate the low-resolution ERG measurement to localized morphological changes. At early stages of the disease, small groups of retinal cells can be in the degenerative or apoptotic forms. Moreover, the ERG provides an integrated signal over whole retinal layers. In principle, ERG a-wave and b-wave reflect outer and inner retinal responses, respectively [21]. However, pathological condition of any individual retinal layer may affect the electrical property for ERG recording, which makes clinical interpretation complicated.
Functional intrinsic optical signal (IOS) imaging [22,23], also termed as optoretinography (ORG) or optophysiology [24], promises a high resolution method for objective assessment of retinal physiology [23]. In analogy to ERG which is based on measurement of stimulus-evoked bioelectric signals, ORG is based on near infrared (NIR) imaging of stimulus-evoked IOS. Stimulus-evoked IOS was first observed in photoreceptor outer segment (OS) suspensions [25][26][27] and isolated retinas [28][29][30]. Recently, functional imaging of stimulus-evoked IOS changes in the retina has been actively explored in both animal and human retinas [22,23,31]. In principle, both stimulus-evoked neural activities and corresponding hemodynamics can contribute to the transient IOS changes in the retina. However, the onset time of fast IOS, which is associated with neural activity, is at millisecond level [32], compared to the second level of hemodynamic associated IOS [33].
Functional IOS imaging of animal models has revealed fast IOS response which occurs almost immediately in the outer retina, i.e. retinal photoreceptors, and delayed IOS changes were also observed in the inner retina with elongated stimulus period [34] or repeated flicker stimuli [22]. Depth-resolved OCT has revealed the OS, which is the center of phototransduction, as the anatomic origin of fast photoreceptor-IOS [32,34,35]. Following studies support that fast photoreceptor-IOS is tightly correlated with the activation phase of phototransduction [36][37][38]. Previous ERG studies have disclosed abnormalities of phototransduction activation in AMD [39][40][41], DR [7,42,43], and RP [44][45][46]. Therefore, the fast photoreceptor-IOS imaging promises a new method for early detection of photoreceptor dysfunction in retinal diseases. However, in vivo IOS imaging of photoreceptor dysfunctions is not yet demonstrated. In vivo verification of fast photoreceptor-IOS distortions in diseased animal models and understanding the pathological mechanism of IOS abnormalities are essential to pave the way toward pursuing clinical deployments of objective ORG of retinal photoreceptors.
In this study, longitudinal in vivo OCT-IOS imaging of wild-type (WT) and retinal degeneration 10 (rd10) mice was conducted to validate ORG detection of photoreceptor dysfunction. Comparative ERG measurement was implemented to characterize the physiological source of fast photoreceptor-IOS. Histological examinations with both light microscopy and electron microscopy were also performed to explore the pathological mechanism of the photoreceptor-IOS distortion caused by retinal degeneration.

Animals
Rd10 (homozygous for the PDE6B rd10 on C57BL/6J background; The Jackson Laboratory, Bar Harbor, ME) was used as a retinal degeneration model, and C57BL/6J (The Jackson Laboratory, Bar Harbor, ME) was used as a control WT model. Rd10 mice exhibit progressive retinal degeneration beginning at around postnatal day (P) 16 at which retinal neurovascular development is in progress [47,48]. Concurrent OCT-IOS imaging and ERG measurements were conducted at P14, P17, P21, and P28. All animal experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were performed following the protocols approved by the Animal Care Committee at the University of Illinois at Chicago.

Experimental setup
A custom-built functional OCT was employed for in vivo retinal IOS imaging. A NIR superluminescent diode (SLD; λ = 850 nm; ∆λ = 100 nm; D-840-HP-I, Superlum Diodes Ltd., Ireland) was used as the OCT light source. Both lateral and axial resolutions were estimated at 3 µm, and 70-kHz A-scan rate was achieved by a linear camera (EV71YEM4CL2014-BA9, Teledyne e2v, England). The linear camera was connected to a frame grabber (PCIe-1429, 680 MB/s bandwidth, National Instruments, Austin, TX) and triggered by a data acquisition board (PCIe-6351, 1.25 MS/s sampling rate, National Instruments, Austin, TX), which also sends trigger output to a galvanometer scanning mirror (GVS001, Thorlabs, Newton, NJ) and a LED driver (LEDD1B, Thorlabs, Newton, NJ) for synchronization. The ERG recording system was also integrated with the OCT system. A silver wire was bent into a circle and placed in contact with the cornea, serving as an active electrode. Another silver electrode was twisted around the bite bar and was in contact with the oral cavity, serving as a reference electrode. The ERG was amplified 1000x and filtered (pass-band: 1 Hz to 10 kHz) using a bioelectronic amplifier (DAM50, World Precision Instruments, Sarasota, FL) and then sampled by the PCIe-6351 data acquisition board. Data acquisition, real-time imaging preview, and system control were done by a custom-designed LabVIEW program (LabVIEW 2013, National Instruments, Austin, TX). Image reconstruction and data processing were done by MATLAB (MATLAB R2016a, MathWorks, Natick, MA). Details of the OCT system are also described in a previous study [37].

Experimental procedure
Each mouse for imaging was fully dark-adapted for 3 hours. The experimental preparation was implemented under a dim red light, and imaging recording was conducted in dark condition. The mouse was first anesthetized by intraperitoneal injection of a mixture of 100 mg/kg ketamine and 5 mg/kg xylazine. A drop of 1% atropine sulfate was applied to the imaging eye for pupil dilation. After the pupil was fully dilated, the mouse was placed onto the animal holder. The active ERG electrode was placed in contact with the cornea. A drop of eye gel was applied on both eyes and then a cover glass was placed on the imaging eye. OCT images were acquired approximately 400 µm nasally away from the optic nerve head (ONH). OCT B-scan consisting of 112 A-lines was acquired with 2 ms temporal resolution. Total recording time per measurement was 3 sec including 1 sec for pre-stimulus and 2 sec for post-stimulus. The ERG was simultaneously recorded. A 10 ms green light generated by a 505 nm LED (M505L3, Thorlabs, NJ) was delivered to the dark-adapted retina. The experiment was performed only once at a time per mouse.

IOS data processing
The IOS data processing has been documented previously [34,49]. Briefly, raw OCT B-scans were first registered to compensate for eye movements using a sub-pixel registration algorithm [34]. The active-IOS pixels were then identified from a sequence of the registered OCT B-scans. Any pixel that markedly changes its value in response to the light stimulus was identified as the active-IOS pixel that can be either positive or negative. To minimize false counting, two conditions were sequentially applied: 1) the three-sigma (3-σ) rule and 2) the consecutive rule, i.e., a pixel changing intensity value greater or less than three standard deviations ± pre-stimulus mean over the consecutive number of the post-stimulus frames is assigned as positive or negative IOS pixel, respectively. For tracing dynamic intensity change, the IOS pixel values in pre-stimulus B-scans were averaged and used as a background intensity image (I) and the background intensity value was sequentially subtracted from each post-stimulus B-scan in a pixelwise manner (∆I).

Sample preparation for light microscopy examination
At each time point, retinal slices from 3 mice were used for histological examination of each strain. The mice were randomly selected from 3 different litters of each strain. Mice were first euthanized by carbon dioxide inhalation, followed by enucleation. The eyeball was placed in a cryomold filled with tissue freezing medium. The mold was fully covered by the freezing medium and then immediately moved onto dry ice. Next, retinal slices near the ONH were cryo-sectioned with 10 µm thickness. Retinal slices were placed on the glass slide and subsequently fixed with a mixture of 4% paraformaldehyde and 1% glutaraldehyde for 20 min. The fixative was washed by 1x PBS and then hematoxylin (ab245880, Abcam, MA) was incubated for 5 min. After that, hematoxylin was rinsed in three changes of distilled water prior to light microscopy.

Sample preparation for electron microscopy examination
Retinal slices from 3 WT and 3 rd10 mice at P17, independent from the IOS/ERG recording group, were used for electron microscopy of photoreceptor OSs. The mice were randomly selected from 3 different litters in each strain. Freshly extracted eyeballs were placed in a cryomold filled with tissue freezing medium and snap frozen on dry ice. The frozen retina was cut by a scalpel into a small rectangle block. The retinal block was fixed with a mixture of 4% paraformaldehyde and 1% glutaraldehyde for more than 48 hours, followed by the secondary fixation with osmium tetroxide as the lipid-rich OS disc structures are not well preserved by aldehydes [50]. Tissue samples were dehydrated in graded series of ethanol: 30%, 50%, 70%, 90%, 95%, and 100%. Next the samples were cut by an ultramicrotome diamond knife (Leica Ultracut UCT; Leica Microsystems, Buffalo Grove, IL) to obtain retinal slices with 70 nm thickness. Afterwards, the samples were stained with alcoholic uranyl acetate and then stained with saturated methanolic uranyl acetate for 5 min and Venable and Coggeshall lead citrate for 5 min prior to observation.

Statistical analysis
14 WT mice from 3 different litters and 22 rd10 mice from 4 different litters were initially used for longitudinal IOS/ERG measurement with a stimulus photon flux (1.88 × 10 7 photons/ms/µm 2 ). For the rd10, as apparent photoreceptor cell losses were confirmed after P21, only 14 mice at P21 and 9 mice at P28 were imaged. Additional 30 WT mice at P28, independent from the longitudinal recording group, were used to examine IOS/ERG responses with 4 different photon fluxes (9.30 × 10 5 photons/ms/µm 2 , 2.81 × 10 6 photons/ms/µm 2 , 5.68 × 10 6 photons/ms/µm 2 , and 1.88 × 10 7 photons/ms/µm 2 ). Data from the group stimulated by 1.88 × 10 7 photons/ms/µm 2 were also included in the longitudinal group analysis for P28 WT as the experimental condition was applied in the same manner. OCT data with severe motion artifacts, which cannot be corrected by registration, and noisy/missing ERG data due to poor contact between the active electrode and the cornea were excluded in the analysis. Specific animal numbers used in statistical analysis are also provided in each figure caption. All data are expressed as mean ± standard error of the mean (SEM) per group. Two independent sample t-tests for WT and rd10 at each age were performed using either student's t-test (equal variances) or Welch's t-test (unequal variances), and p-value less than 0.05 was considered statistically significant. Figure 1 shows representative functional OCT-IOS imaging of an adult WT mouse. The depth-resolved OCT can clearly differentiate individual retinal layers ( Fig. 1(A)). A 10 ms flash (central wavelength: λ = 505 nm; photon flux: 1.88×10 7 photons/ms/µm 2 ) was applied to the dark-adapted retina for simultaneous OCT-IOS imaging ( Fig. 1(B)) and ERG measurement ( Fig. 1(D)).

OCT-IOS imaging and ERG measurement
It was observed that fast photoreceptor-IOS was predominantly confined within the OS region and occurred almost immediately after the stimulus onset. In other words, potential signal contaminations from slow IOS change due to inner neural activities or hemodynamic changes can be omitted for the 100 ms post-stimulus recording period ( Fig. 1(B)), consistent to our previous observations [32,37]. The number of active-IOS pixels was counted within the outer retina ( Fig. 1(C1)), i.e. the region between external limiting membrane (ELM) and Bruch's membrane (BM), in which the fast photoreceptor-IOS response occurs. The active-IOS pixels can be further classified as either positive (increasing) or negative (decreasing) IOS changes (Figs. 1(B) and 1(C)). Corresponding IOS magnitude change was also quantitatively evaluated ( Fig. 1(C2)). Figure 1(C) confirmed that fast photoreceptor-IOS response occurred within 2 ms from the stimulus onset and reached the peak-magnitude within 20 ms. Therefore, a 20 ms post-stimulus recording was selected for photoreceptor-IOS characterization of WT and rd10 in the following sections.  Fig. 2(B). At P14, there was no significant difference observed between WT (31.8 ± 11.3) and rd10 (29.3 ± 9.8). The retina itself was naturally immature at P14. However, a large discrepancy was disclosed at P17 (79 ± 9.8 in WT; 25.9 ± 9.4 in rd10; P < .001). The enhanced photoreceptor-IOS response reflects normal photoreceptor development in WT, while rd10 manifested degraded IOS response with the onset of photoreceptor degeneration [48,51]. Figure 2(C) shows temporal profiles of intensity variation of active-IOS pixels. The photoreceptor-IOS occurred rapidly within 2 ms after the stimulus onset and gradually reached a plateau within 20 ms (Fig. 2(C1)). Notably, as shown in Fig. 2(C3), the peak IOS intensity in rd10 was significantly reduced due to photoreceptor degeneration (P21: 1.01 ± 0.03 in WT and 0.73 ± 0.07 in rd10 [P < .01]; P28: 1.01 ± 0.02 in WT and rd10 0.55 ± 0.07 [P < .001]). These results demonstrate that fast photoreceptor-IOS is highly sensitive to photoreceptor viability as well as structural maturity.

Spatial characteristics of fast photoreceptor-IOS
To further characterize spatial distribution of fast photoreceptor-IOS, density maps of active-IOS pixels from WT dataset were constructed within the outer retinal region (ELM to RPE). As shown in Fig. 3(A), the active-IOS pixels were consistently mapped along the interdigitation zone (IZ) and the RPE that are directly in contact with the rod OS tips. The IZ and RPE represent hyperreflective zones with granular texture, and a notable change of this granular texture upon a light stimulus is recognized in a sequence of OCT B-scans (see Visualization 1). Changing granular texture means rearrangement of pixel values between interframes, which ultimately contributes to the generation of active-IOS pixels. We also quantified the global impact of fast photoreceptor-IOS on OCT signals of the entire outer retinal area. Temporal intensity profiles of the entire outer retina show that the overall intensity value was barely changed over time in response to the light stimulus ( Fig. 3(A2)). In addition, an M-scan, referring to consecutive averaged A-scans (central 60 out of 112 A-lines per B-scan) at the same lateral position, and corresponding OCT signal profiles in the direction of A-scan before and after a stimulus are constructed in Fig. 3(B). It shows that neither the ELM nor BM band has detectable position shift while the fast photoreceptor-IOS response occurs. These results suggest that the fast IOS is a local event within the OS-RPE complex. Given that the RPE alone does not evoke the fast IOS, the OS is the only triggering source of fast photoreceptor-IOS. This observation also confirms that high spatial resolution is essential to ensure the sensitivity of robust IOS imaging [52]. Without necessary spatial resolution, the IOS changes with opposite polarities, i.e. positive and negative changes, can be pooled to each other in adjacent volumes.

Histological analysis of photoreceptor degeneration
To better correlate IOS anomaly with retinal structural change in rd10, we examined morphological features of the retina. Significant retinal thinning was first confirmed in rd10 due to progressive photoreceptor degeneration (Fig. 4(A2)). Photoreceptor shortening was a histological hallmark of the onset of photoreceptor degeneration [53]. Besides, OCT images reveal an abnormal hyperreflective zone appeared in the photoreceptor (yellow arrow in Fig. 4(A2)). In addition, the retinal detachment was occasionally observed in P28 rd10 (Fig. 4(A2)). Length of photoreceptor inner segment (IS) and OS was also measured from ELM to RPE. The RPE layer was included due to unclear boundary at the OS tip. In WT, the photoreceptor length continued to increase with age ( Fig. 4(B)), and P14 length was similar between the two genotypes (P = .194), confirming that morphological structure of rod photoreceptors is intact in rd10 at the time mouse eyes open ( Fig. 4(B)) [53,54]. To demonstrate the rationale of the diffusive hyperreflective zone and IOS anomaly observed in the OS of P17 rd10, electron microscopy was performed. P17 WT shows tightly stacked and well aligned OS discs (Fig. 4(C)); however, electron micrographs of rd10 at P17 reveals the OSs were highly disorganized and fragmented (Fig. 4(D1)). Enlarged disc membranes and empty disc spaces were observed along the OS body (Fig. 4(D2)). In addition, a unique abnormal feature shown as swirls of disc membrane was frequently observed (Fig. 4(D3)). These results demonstrate that ultrastructural abnormalities of the OS discs initially occur in rd10 and accompany the following rod cell death, and this transition moment is captured by OCT as the diffusive hyperreflective zone in the OS (Fig. 4(A2)). Electron microscopy observation further indicates that disrupted OS discs have a direct impact on fast photoreceptor-IOS.

Comparative photoreceptor-IOS recording and ERG measurement
To further investigate physiological mechanism of fast photoreceptor-IOS, comparative ERG measurement of WT and rd10 was implemented. At P14, both ERG a-and b-wave amplitudes were comparable between WT (a-wave: 0.19 ± 0.02 mV; b-wave: 0.26 ± 0.02 mV) and rd10 (a-wave; 0.19 ± 0.01 mV; b-wave; 0.26 ± 0.01 mV). However, the implicit time of a-wave was significantly delayed in rd10 as shown in the inset of Fig. 5(A1) (WT: 6.98 ± 0.1 ms; rd10: 13.93 ± 0.84 ms; P < .001). ERG of rd10 has been found to be never normal [55,56], and we found that the early abnormality is indicated as the delayed implicit time. In addition, the a-wave of rd10 rapidly diminished with age due to rod cell degeneration (P21: 0.06 ± 0.01 mV) and was not detectable at P28. In contrast, the b-wave of rd10 maintaining a certain level of amplitude (P14: 0.26 ± 0.01 mV; P17: 0.23 ± 0.02 mV; P21: 0.26 ± 0.02 mV; P28: 0.13 ± 0.02 mV) demonstrates that the inner retinal circuitry is relatively resistant to the rod photoreceptor degeneration [48,57]. The surging b/a wave ratio of rd10 well reflects this context as well (Fig. 5(B4)). In WT, the amplitude of both a-and b-wave became higher with retinal development and reached the upper bound at P21 (a-wave: 0.38 ± 0.03 mV; b-wave: 0.68 ± 0.05 mV) [58]. The implicit time of ERG a-wave in WT was merely changed with age (P14: 6.98 ± 0.1 ms and P28: 6.95 ± 0.15 ms).
Intriguingly, unlike ERG a-wave peaked at P21, the active-IOS pixel number in P28 WT was significantly larger than that in P21 WT (Fig. 2(B3); 196.6 ± 28.6 at P21; 305.8 ± 31.5 at P28; P < .05). In addition, Fig. 6(A) shows that the time course of fast photoreceptor-IOS clearly precedes that of ERG a-wave. Most of the active-IOS pixels emerged within 4 ms after the onset of light stimulus, while the a-wave reached only ∼39% of its maximum response at 4 ms ( Fig. 6(A)). This observation confirms that the physiological source of fast photoreceptor-IOS precedes that of hyperpolarization, i.e. the closing of cyclic GMP-gated channels in the OS membrane [36,53].
To further examine signal characteristics of the photoreceptor-IOS and ERG a-wave, light stimuli with four different photon fluxes were used to activate the WT retina at P28 (Fig. 6(B)). Although the implicit time of ERG a-wave was delayed corresponding to reduced flash strength [59,60], all light conditions accompany similar ERG a-wave amplitudes as a result of biochemical amplification of phototransduction cascade (Fig. 6(B1)) [61]. On the other hand, the magnitude of photoreceptor-IOS response has a direct correlation with the number of incident photons (Fig. 6(B2)), demonstrating that the sensitivity of fast photoreceptor-IOS has a close relationship to rhodopsin activation. Taken together, these results demonstrate that the photoreceptor-IOS response is highly dependent on the photon flux and initiates at the activation phase of phototransduction.

Discussion
In this study, longitudinal OCT-IOS imaging and ERG measurement of WT and rd10 mice were conducted to validate in vivo ORG detection of photoreceptor dysfunction due to retinal degeneration. Depth-resolved OCT revealed fast photoreceptor-IOS at the OS region within 2 ms from the stimulus onset. The fast photoreceptor-IOS has no significant difference between WT and rd10 at P14, while three-times magnitude difference of the active-IOS pixels was observed at P17. This observation indicates that fast photoreceptor-IOS is remarkably sensitive to photoreceptor dysfunction at the early stage of retinal degeneration. Comparative ERG measurement confirmed that the response time of fast photoreceptor-IOS is clearly ahead to the a-wave, and electron micrographs revealed disrupted OS discs in the rd10 as the pathological origin of fast photoreceptor-IOS abnormality.
In rd10 mice, the fast photoreceptor-IOS gradually degraded due to retinal degeneration. In WT mice, the fast photoreceptor-IOS continued to grow with retinal development, indicating a direct correlation to photoreceptor maturity. Rod OSs are known to be the last structures to develop in the mammalian retina, and the ultrastructure of rodent OSs becomes fully mature at 4 to 6 weeks of age [62][63][64]. This is consistent with our hypothesis that fast photoreceptor-IOS primarily attributes to transient OS conformational changes elicited by visible light stimulation. Previous studies have demonstrated light induced changes of OS disc morphology [47,[65][66][67][68][69]. Stimulus-evoked transient OS conformational change has been observed in freshly isolated retinas [35,36,70,71] and intact animals [36,72]. Comparative electron microscopy of darkand light-adapted retinas disclosed light-driven shrinkage of inter-disc space of OS [73]. With pharmacological treatment, a recent study demonstrated that the stimulus-evoked OS changes occur before the hyperpolarization of retinal photoreceptors [36], which is consistent with the observation that fast photoreceptor-IOS occurs before PDE activation [53]. Similarly, recent study revealed a rapid reduction of optical path length in human cone OSs [74,75], which might reflect stimulus evoked OS shrinkage.
Our study provides three evidences to support that fast photoreceptor-IOS response attributes to transient OS conformational change due to visible light stimulation. First, the distribution maps confirmed the OS-RPE complex as the anatomic location of fast photoreceptor-IOS (Fig. 3(A1)). It was observed that the active-IOS pixels were primarily confined within the region between the OS tips and RPE. This region has no anatomic junctions bridging the gap and is vulnerable to physical impacts as the retinal detachment observed in rd10 [76]. Instead, long thin microvilli on the RPE apical surface support an adhesion of the OS on the RPE by ensheathment, which can allow the OS tips to be relatively flexible [76]. Moreover, we can rule out significant scattering property changes by the OS compartments during the phototransduction cascade, given the fact that the OS body remains as a hyporeflective OCT band ( Fig. 3 and Visualization 1). Second, the consecutive OCT B-scans revealed a rapid rearrangement of pixel values in the OS tips, IZ, and the apical surface of RPE (Visualization 1), which gives rise to likelihood of a sudden physical intervention on that region. The OSs might have waveguide property [77,78], and thus conformational change of the OSs may directly affect the image pattern. Third, the number of active IOS pixels increased with photoreceptor development in WT mice (Fig. 2(B)). Considering the OS conformational change is a contractile shift [72], the OS tips would slightly pull up the RPE apical surface, which results in a little extension of the IZ/RPE hyperreflective zones toward the inner retina. As the OSs develop with age, this extension may become rigorous, generating more active IOS pixels.
Comparative ORG and ERG measurements clearly revealed distinct characteristics of the fast photoreceptor-IOS from ERG a-wave. First, our data show that the ERG a-wave in WT reached the peak at P21 (Fig. 5(B1)), and the rod photoreceptors matured in size at P21 as the photoreceptor length (ELM to RPE) increased only ∼4% from P21 to P28 (Fig. 4(B)). This result agrees with a previous observation that the amplitude of a-wave is proportional to the OS length [62]. In contrast, the number of active-IOS pixels increased ∼60% from P21 to P28 (Fig. 2(B3)). This major difference of the photoreceptor-IOS between P21 and P28 cannot be explained solely by the factor of OS length. Instead, immature inner components of the rod OS at P21, such as disc organization and rhodopsin contents [62,64], may directly contribute to the IOS response. Considering the cycle of disc renewal rate (1.8 to 2.2 µm/day in mice) [79], a diurnal event in which small packets of discs are newly synthesized from the IS and pruned from the distal end of the OS, WT at P21 should still hold immature discs in the distal OSs, which synthesized even 10 days ago. Second, by virtue of amplification of the phototransduction cascade, a single photon can close 5% of rod cyclic GMP-gated channels on the plasma membrane [80], and about 100 photoactivated rhodopsins are enough to cause half-saturated a-wave [81]. However, this extremely low level light limits the photoreceptor-IOS response (Fig. 6(B2)), and a dynamic range of the photoreceptor-IOS response is much larger than that of a-wave amplitude to variable stimulus intensities [37]. Moreover, the response time of fast photoreceptor-IOS is clearly ahead to the a-wave. Our results show that ∼88% of the maximum photoreceptor-IOS response was achieved within 4 ms, but the a-wave reached only ∼39% of its maximum amplitude at 4 ms ( Fig. 6(A)). A previous study also reported that the onset times of the fast photoreceptor-IOS and a-wave were 1.1 ± 0.2 ms and 5.0 ± 0.5 ms, respectively, in adult WT mice [37]. Other studies further confirmed that onset times of the OS shrinkage occurred before the hyperpolarization of photoreceptors [36,53].

Conclusion
Longitudinal OCT-IOS imaging of WT and rd10 has demonstrated the feasibility of objective ORG detection of photoreceptor dysfunction due to retinal degeneration. Depth-resolved OCT imaging and analysis confirmed the transient OS change as the physical source of fast photoreceptor-IOS. Comparative ORG and ERG measurement verified the activation phase of phototransduction as the physiological correlator of the fast photoreceptor-IOS, which may provide a unique biomarker for early detection of photoreceptor dysfunction. High resolution electron microscopy revealed disorganized OS discs, i.e. the pathological origin of the photoreceptor-IOS distortion. We anticipate that further development of fast photoreceptor-IOS imaging will advance objective ORG diagnosis and treatment assessment of AMD, RP, and other diseases that can cause photoreceptor dysfunctions.