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Multidisciplinary Ophthalmic Imaging  |   October 2012
Stimulus-Specific Pupil Dynamics Measured in Birds (Gallus gallus domesticus) In Vivo with Ultrahigh Resolution Optical Coherence Tomography
Author Affiliations & Notes
  • Alireza Akhlagh Moayed
    From the Department of Physics and Astronomy, the
  • Vivian Choh
    School of Optometry and Vision Science, and the
  • Sepideh Hariri
    From the Department of Physics and Astronomy, the
  • Chenyi Liu
    Systems Design Engineering Department, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada.
  • Alexander Wong
    Systems Design Engineering Department, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada.
  • Kostadinka Bizheva
    From the Department of Physics and Astronomy, the
  • Corresponding author: Kostadinka Bizheva, Department of Physics and Astronomy, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada N2L 3G1; kbizheva@uwaterloo.ca
Investigative Ophthalmology & Visual Science October 2012, Vol.53, 6863-6869. doi:10.1167/iovs.12-10291
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      Alireza Akhlagh Moayed, Vivian Choh, Sepideh Hariri, Chenyi Liu, Alexander Wong, Kostadinka Bizheva; Stimulus-Specific Pupil Dynamics Measured in Birds (Gallus gallus domesticus) In Vivo with Ultrahigh Resolution Optical Coherence Tomography. Invest. Ophthalmol. Vis. Sci. 2012;53(11):6863-6869. doi: 10.1167/iovs.12-10291.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: To demonstrate the ability of high speed, ultrahigh-resolution optical coherence tomography (UHR-OCT) to measure and characterize in vivo visual stimulus-specific pupil dynamics in birds.

Methods.: Ten two-week old White Leghorn ( Gallus gallus domesticus ) chickens were imaged in this study. The chickens were dark-adapted for 1 hour and anesthetized with 2% isoflurane prior to the imaging procedure. Blue, green, and red single flash visual stimuli of 7 ms duration were used to evoke pupillary responses. UHR-OCT cross-sectional images of the pupil were acquired prior, during, and for several seconds after the visual stimuli onset. Images were processed with a novel custom automatic algorithm, designed to determine the pupil diameter changes over time.

Results.: Results from this study show that the pupillary constriction begins with the onset of the visual stimuli; however, maximum pupil constriction occurs ∼150 ms later. No statistically significant variation in the timing of the maximum pupillary constriction was observed for stimuli of different colors. However, significant variation was observed in the maximum pupil constriction amplitudes, between red-green and red-blue stimuli, but not between blue-green stimuli. Furthermore, the magnitude of the maximum pupil constriction decreased monotonically with time under isoflurane anesthesia.

Conclusions.: We demonstrated, for the first time, measurements of visually evoked pupillary dynamics in animals using high speed UHR-OCT. The results suggest dependence of the pupillary dynamics on the color of the visual stimulus, and adverse effects of isoflurane anesthesia on the visually evoked pupillary responses in chickens.

Introduction
Optical coherence tomography (OCT) is a well-established optical method for noninvasive, cellular level resolution imaging of biological tissue. 1 Over the past decade, ultrahigh-resolution OCT (UHR-OCT) has been used for fast, volumetric, morphological imaging of healthy and diseased retina 2,3 and cornea. 4 In addition to imaging the structure, UHR-OCT is able to image blood perfusion 5 and quantify blood flow, 6 provide information about the birefringent properties, 79 and even measure visually evoked physiological responses in ocular tissues. 1016  
Recently, our research group demonstrated in vivo imaging of visually evoked physiological changes in all layers of the chicken retina 17 by use of a modified, functional UHR-OCT system. 18 Since chickens have skeletal intraocular muscles that are not affected by commonly used (smooth muscle) mydriatics—and vecuronium bromide, a skeletal muscle mydriatic, clouds the cornea significantly enough to prevent imaging of the chicken retina with UHR-OCT—only dark-induced natural pupillary dilation was used in those experiments. Because stimulus-induced pupillary constriction may have partially obstructed the optical imaging beam during those experiments, it is possible that the data we recorded is a convolution of visually evoked retinal and pupillary responses. Therefore, in order to isolate the pure stimulus-evoked functional response from the retina, we need to measure the pupillary response to the same stimulus and under the same imaging conditions. 
A number of studies were conducted in the past to investigate visually evoked pupillary responses in birds and avian species in general. 1923 Results from these studies suggest that the maximum pupil constriction occurs on the time scale of hundreds of milliseconds after the stimulus onset and is dependent not only on the color, duration, and intensity of the visual stimulus, but also on the background illumination and the use or absence of anesthesia. Therefore, in order to be able to deconvolve the retinal and pupillary visually evoked responses in our experiments, we need to measure the pupillary dynamics under the same experimental conditions we used for imaging the physiological changes in the chicken retina. 
In this study, we used the same functional UHR-OCT system and visual stimulator used in our functional retina studies 17,18 and modified slightly only the OCT imaging probe to allow for high-resolution morphological imaging of the chicken pupil and iris. The animal handling and imaging procedures remained the same as in the case of the functional retina study, to allow for fair comparison of the results. Here, were present—to the best of our knowledge—the first in vivo measurements of visually evoked pupillary dynamics in domestic chickens acquired with UHR-OCT technology. 
Materials and Methods
Imaging System
Briefly, a high speed (92,000 A-scans/second), research-grade, spectral domain UHR-OCT system (Fig. 1), operating in the 1060-nm wavelength region was used in this study. The choice of imaging in the near-infrared (NIR) wavelength region was made to prevent any pupillary response triggered by the imaging beam. Details about the design and technical specifications of the imaging system were published recently. 18 The UHR-OCT system provided 11-μs time resolution per A-scan and a signal-to-noise ratio (SNR) of ∼97 dB for 1.5 mW power of the imaging beam incident on the cornea (Fig. 1). The original imaging probe designed for retinal imaging consisted of three NIR achromat doublet lenses (Edmund Optics, Barrington, NJ) and a pair of galvanometric scanners (Cambridge Technologies, Bedford, MA). However, for this study, one of the lenses was removed to allow for focusing the UHR-OCT imaging beam at the pupil plane and imaging the chicken iris and pupil with high spatial resolution (3.5 μm axial and ∼30 μm lateral). The eye imaging probe was integrated with a custom multicolor visual stimulator, designed to project an image of the stimulus LED at the pupil plane and generate a Maxwellian view spot of ∼5 mm2 at the chicken retinal surface. 18 The visual stimulator was interfaced to a commercial ERG system (Diagnosys LLC, Lowell, MA), which allows for user-defined selection of the color, duration, and intensity of the visual stimulus. In this study, visual stimuli of blue (455 nm, ∼5 cd/cm2/second, corresponding to 1.66 × 1016 photons/cm2/second); green (530 nm, 218 cd/cm2/second, corresponding to 8.4 × 1017 photons/cm2/second); or red (647 nm, 293 cd/cm2/second corresponding to 1.35 × 1018 photons/cm2/second) colors, and 7 ms duration were used. 
Figure 1. 
 
Schematic of the combined functional UHR-OCT and ERG system with modified imaging probe for imaging the pupil dynamics in chickens: CL, collimating lens; DC, dispersion compensation unit; FC, fiberoptic coupler; M, mirror; NDF, neutral-density filter; PC, polarization controllers; and TS, translation stage. The spectrometer consists of a CL, a volumetric diffraction grating (DG), a focusing lens (FL), and a high-speed InGaAs camera (photodiode array, PDA). The stimulator consists of four color LEDs (white [W], green [G], red [R], and blue [B]), achromat lenses (L1, L2, L3, and L4) and a pellicle (P).
Figure 1. 
 
Schematic of the combined functional UHR-OCT and ERG system with modified imaging probe for imaging the pupil dynamics in chickens: CL, collimating lens; DC, dispersion compensation unit; FC, fiberoptic coupler; M, mirror; NDF, neutral-density filter; PC, polarization controllers; and TS, translation stage. The spectrometer consists of a CL, a volumetric diffraction grating (DG), a focusing lens (FL), and a high-speed InGaAs camera (photodiode array, PDA). The stimulator consists of four color LEDs (white [W], green [G], red [R], and blue [B]), achromat lenses (L1, L2, L3, and L4) and a pellicle (P).
Animal Preparation
Ten two-week old White Leghorn ( Gallus gallus domesticus ) chickens were used for this study (four chickens for blue flash stimulation and three chickens for green and red flash stimulation each). The imaging was conducted in the Biomedical Imaging Lab at the University of Waterloo, with approval from the University of Waterloo animal ethics committee and in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Chickens were dark-adapted for 1 hour prior to the imaging procedure to induce natural pupillary dilation, and then anesthetized with ∼2% isoflurane. The amount of isoflurane anesthesia varied slightly from animal to animal, though it remained unchanged for the duration of each imaging experiment in individual animals. The animals were then placed in a custom animal holder that restricts the head and body motion during the imaging procedure and allows for easy alignment of the eye with respect to the imaging beam. The anesthesia and preparation of the birds for imaging was carried out in a dark room under faint red illumination, while the imaging procedure was carried out in a custom-built dark box, to prevent accidental ambient light exposure of the retina. A lid retractor was used to restrict the motion of the outer eyelid and eye drops (Refresh Tears; Allergan, Irvine, CA) were administered frequently to hydrate the cornea in order to keep it optically transparent. 
Data Acquisition and Image Processing
The eye imaging probe was positioned so that the UHR-OCT imaging beam is focused at the pupillary plane of the chicken eye, where an image of the visual stimulator LED is formed. The UHR-OCT imaging beam was also aligned so that it is perpendicular to the apex of the cornea, thus creating a high-intensity specular reflection artefact along the optical axis of the eye in the UHR-OCT cross-sectional tomograms. Images were acquired at the maximum camera speed (92,000 A-scans/second) and the alignment of the imaging probe was frequently checked and adjusted when necessary, to ensure that the UHR-OCT cross-sectional images were acquired along the pupil diameter plane. To measure the visually evoked pupil diameter changes, a series of functional UHR-OCT recordings were acquired from the chicken iris and pupil, each recording consisting of 720 B-scans (2D cross-sectional images of the iris and pupil). Each B-scan consisted of 512 A-scans and corresponded to acquisition time of ∼6.7 ms. The total time for each functional UHR-OCT recording, was ∼5 seconds. The first 74 B-scans of each functional UHR-OCT recording were acquired in darkness followed by a 7-ms flash of the chosen color. The rest of the recording was completed in darkness. 
UHR-OCT images were generated from the raw data using a custom statistical code (MATLAB; MathWorks, Natick, MA). A novel algorithm was developed to identify the pupil edges from the UHR-OCT images, track their movement over time, and calculate the pupil diameter from the spatial coordinates of the pupil edges. The algorithm is based on the following steps: noise-compensated reconstruction for generation of speckle-suppressed UHR-OCT images; identification and tracking of the pupil edges in successive UHR-OCT image frames, by use of region-based maximum likelihood tracking strategy; and calculation of the pupil diameter from the spatial coordinates of the pupil edges and tracking the pupil size changes over time. The new algorithm was also implemented using statistical software (MathWorks). 
Results
Figure 2A shows a representative B-scan from the chicken pupil with the edges of the pupil outlined by the highly reflective tissue of the iris. The black vertical line in the center of the image marked with the black arrow is caused by the strong specular reflection of the imaging beam from the surface of the corneal apex, which is used as a marker to align the pupil with respect to the imaging beam. A faint reflection of the chicken lens surface is also visible on the image (gray arrow). Figure 2B shows the same image with marked edges of the pupil, where the pupil diameter is marked with a gray arrow. Figure 2C is a representative movie showing the response of the chicken pupil to blue-light stimulation. 
Figure 2. 
 
A representative cross-sectional UHR-OCT image of the chicken iris and pupil. (A) The gray arrow marks the reflective surface of the eye lens, while the black arrow marks the imaging artefact generated by the strong back-reflection of the imaging beam off the corneal apex. (B) The same tomogram with a gray arrow pointing at the pupil edges as determined automatically by the novel pupil edge detection algorithm. (C) A movie of the pupil dynamics.
Figure 2. 
 
A representative cross-sectional UHR-OCT image of the chicken iris and pupil. (A) The gray arrow marks the reflective surface of the eye lens, while the black arrow marks the imaging artefact generated by the strong back-reflection of the imaging beam off the corneal apex. (B) The same tomogram with a gray arrow pointing at the pupil edges as determined automatically by the novel pupil edge detection algorithm. (C) A movie of the pupil dynamics.
 
Figure 2C (movie)
For all birds, a minimum of five recordings of the change in the pupil size as a function of time were acquired for a particular color. Statistical results obtained from multiple recordings acquired from a single bird are shown in Figures 3A–3C for 7 ms for blue (12), green (8), and red (5) visual stimuli, respectively. Data shown in the graphs were normalized relative to the initial pupil diameter and then averaged over multiple recordings from the same chicken. No standardization was applied with respect to maximum pupillary response for stimuli with different colors. In general, the pupillary responses between multiple flashes of the same color were relatively consistent for the constriction phase, but dilation of the pupil showed slightly more variability with time. 
Figure 3. 
 
Mean and SEM of the fractional pupil size changes as a function of time for multiple recordings obtained from one animal (A) blue, (B) green, (C) red light stimuli of the same duration. (DF) Comparison of mean pupil dynamics recordings acquired from different animals for the three colors of the visual stimulus. (GI) Mean and SEM of the fractional pupil size changes as a function of time for all chickens for the three colors of the visual stimulus.
Figure 3. 
 
Mean and SEM of the fractional pupil size changes as a function of time for multiple recordings obtained from one animal (A) blue, (B) green, (C) red light stimuli of the same duration. (DF) Comparison of mean pupil dynamics recordings acquired from different animals for the three colors of the visual stimulus. (GI) Mean and SEM of the fractional pupil size changes as a function of time for all chickens for the three colors of the visual stimulus.
Figures 3D–3F show individual pupillary responses of the birds to blue, green, and red light stimuli, respectively, without normalization of the maximum pupillary constriction. For all wavelengths used, maximum pupillary constriction amplitudes average to ∼10% of the pupil (range: ∼4% to ∼16%) and occurs ∼160 ms (range: 100–225 ms) after the stimulus onset. Pupillary dilation dynamics subsequent to the constriction follows two general trends, one where the pupil dilates fairly fast and reaches a steady state (plateau) ∼1 second post-stimulus, although it may or may not return to the prestimulus size, or one where the pupillary dilation occurs much more slowly, and pupils do not recover their original sizes until well after ∼4.5 seconds post-stimulus. Of the three colors, the red flash appears to elicit the most consistent response in terms of dynamics (Fig. 3F), with all birds exposed to this color showing the slower dilation response. In contrast, three of the four birds exposed to the blue flash show the “plateau” response (Fig. 3D), while responses to the green light (Fig. 3E) show the greatest variation, with dilation rates alternating between the plateau and slow responses. 
Figures 3G–3I summarize results from a statistical analysis carried out on all functional UHR-OCT recordings acquired from all chickens used in this study. Not surprisingly, the mean pupillary responses for the red and blue flashes resemble their components, with the average response to the blue flash showing a plateau (Fig. 3G) and the average response to the red flash showing the slower dilation response (Fig. 3I). The mean response to the green flash shows a tail-end rate of dilation that lies between those for the blue and red flashes (Fig. 3H). 
The dilation responses for all colors appear to be at least biphasic, showing an initial quicker dilation, followed by a slower return to initial pupil size (Fig. 4A; summary of Figs. 3G–3I). The responses from individual birds (Figs. 3D–3F) appear to confirm that two phases of pupillary dilation exist for almost all the tested birds. On the assumption that at least two of the phases are “real,” the slope of the initial linear phase (S1 on Fig. 4A) was calculated and is presented in Fig. 4C. The initial fast dilation rate is greatest for the red and smallest for the green stimulus. In contrast, the second phase of dilation is slowest for the blue, as it reaches its plateau relatively quickly and develops differently over time compared to stimuli of different color. Therefore, the second pupillary dilation phase was fitted by a proper nonlinear function for each color of the visual stimulus (Fig. 4A). It is clear from Figure 4A that the constricted pupil dilates exponentially in the case of blue stimuli, linearly for green stimuli, and reciprocally in the case of red stimuli. 
Figure 4. 
 
Statistical analysis of the pupillary dynamics data. (A) The average recordings of the fractional pupil size change as a function of time for all chickens for blue, green, and red color flashes and their fitted functions. (B) Slope of the first phase of dilation (S1) for all three colors. (C) Statistical summary from ∼90 pupil dynamics recordings from 10 chickens for the maximum pupil constriction and its latency with respect to the onset of the visual stimulus for the blue, green, and red color stimuli.
Figure 4. 
 
Statistical analysis of the pupillary dynamics data. (A) The average recordings of the fractional pupil size change as a function of time for all chickens for blue, green, and red color flashes and their fitted functions. (B) Slope of the first phase of dilation (S1) for all three colors. (C) Statistical summary from ∼90 pupil dynamics recordings from 10 chickens for the maximum pupil constriction and its latency with respect to the onset of the visual stimulus for the blue, green, and red color stimuli.
It is clear from Figure 4A that red flashes also result in maximal constriction (mean ± SEM: 9.5% ± 1.1% change in pupil diameter), while blue and green flashes elicit minimal constriction (5.5% ± 0.6% and 7% ± 0.5%, respectively) of the chicken pupil. The maximum pupil constriction and its latency were calculated from ∼90 pupil dynamics recordings acquired from 10 chickens with the blue, green, and red color stimuli. A statistical summary of the data is presented in Figure 4B, where the yellow vertical line marks the timing and duration of the visual stimulus. Pupillary constriction amplitudes are significantly different depending on the color of the stimulus used (P = 0.003), with amplitudes in response to a red flash significantly greater than for the blue (P = 0.003) and green (P = 0.031) flashes. The amplitudes for the blue and green flashes were not different (P = 0.765). The latencies of the constrictions were not different depending on color (P = 0.297). 
The large variability of the pupil responses to the same color, intensity, and duration of the visual stimuli observed across different animals in the study suggested that anesthesia may have some effect on the pupillary dynamics. Analysis of the pupillary responses from chickens of the same age and sex to light stimuli of the same color, intensity, and duration at different times from onset of the same levels of isoflurane anesthesia revealed a decrease in the pupillary constriction magnitude with prolonged exposure to isoflurane anesthesia (Fig. 5). 
Figure 5. 
 
The effect of isoflurane anesthesia on the pupil constriction and subsequent dilation measured at different time points between 5 and 40 minutes relative to the onset of the anesthesia for blue (A), green (B) and red (C) color of the visual stimulus.
Figure 5. 
 
The effect of isoflurane anesthesia on the pupil constriction and subsequent dilation measured at different time points between 5 and 40 minutes relative to the onset of the anesthesia for blue (A), green (B) and red (C) color of the visual stimulus.
Discussion
Our results show that visually evoked pupillary constriction begins almost simultaneously with the stimulus onset. Our latency responses for peak pupillary constriction were approximately 3 times slower than those observed by Barbur et al. 20 However, there are many differences in the paradigms between our studies, including but not limited to: stimulus duration (7 ms versus 500 ms); intensity and projection of the stimulus (spatial frequency and focal plane); age of the chicken; and pupil size at the start of the experiment. Moreover, our chickens were raised in artificial light, while those of Barbur's group 20 were raised in natural sunlight, which could have an effect on the spectral sensitivity of the chicken as well as other effects on pupil dynamics. 
While our method provides a novel way for monitoring and characterization of pupillary dynamics, it should be acknowledged that it is sensitive to noise and variability. Iridial constriction amplitudes were clearly affected by the amount and duration of anesthesia and our method is also sensitive to eye motion; although animals were anesthetized and the head motion was restricted, translation and rotation of the chicken eyeball during the imaging procedure induced some uncertainty in the measurement of the pupil diameter, which may partially account for some of the variability that was observed in our data (Figure 3G–3I). 
Due to the integrated design of the OCT imaging probe and the visual stimulator, necessary to provide stability by coaxial alignment of the imaging and stimulus beams, only one eye could be measured and data for consensual pupillary responses were not available. Potentially, a second visual stimulator can be interfaced to the OCT+ERG system to allow for measurement of contralateral pupillary responses. However, building a binocular system comes with other challenges, including but not limited to aligning and keeping the position of the stimulus LED image in the center of the pupil and at the pupil plane in the presence of eye motions. 
Despite the variability mentioned above, we were able to detect wavelength-dependent differences, with the pupillary constriction amplitudes to red stimulation significantly greater than either of the other two colors. At least some of the differences must be related to the relative intensities of the flashes used, with the red flash having the highest intensity and the blue the lowest. However, it should also be noted that other investigators have found that red flashes of light are behaviorally preferred, 24 and induce stronger pupillary constriction amplitudes 20 and ERG signals. 25  
Presumably, the different-colored flashes were detected by the visible spectrum-sensitive cones, of which chickens possess three, long wavelength-sensitive (red) cones, medium wavelength-sensitive (green) cones, and short wavelength-sensitive (blue) cones. Chickens are tetrachromats, that possess ultraviolet (UV)-sensitive cones, and the spectral sensitivities of the chicken cones are further complicated by the presence of six types of oil droplet filters, which, in general, act to fine-tune the spectral sensitivity of the cone in which they are embedded. 26 Several investigators reported relatively similar peak-wavelength sensitivities of the four cones, with slight variations primarily dependent upon whether peak sensitivities were measured using electrophysiological or psychophysical means and whether oil droplet filtering has been taken into account. The sensitivities range from 562 to 602 nm, 507 to 540 nm, 455 to 475 nm, and 413 to 420 nm, for the longest to shortest wavelength-sensitive cones, respectively. 2630  
In the chicken, melanopsin-containing, intrinsically photosensitive retinal ganglion cells (ipRGCs) are localized to two regions in the retina: the retinal ganglion layer and the outer regions of the inner nuclear layer. 31,32 As noted for ipRGCs in other species, the chick ipRGC sensitivities are maximal in the blue range, with an absorption maximum at 468 nm and the range of response maxima between 402 and 473 nm. 33 In humans, blue light-induced pupillary constriction is more stable 34 and is greater in amplitude 35,36 than that for red light when matched for photopic luminance. Blue light-exposed ipRGCs mediate circadian rhythms and pupillary light reflexes in GUCY1* chickens, a blind breed of birds that do not have functional rods or cones. 23 It is likely that ipRGCs contributed to the blue flash response in our study, but because of the disparity in the light flash intensities, their contribution remains unknown. 
With the possible exception of one bird (Fig. 3E), our short (7 ms) flash was not of sufficient duration to allow for a sustained contracture of the iris muscles, resulting in a biphasic dilation of almost all pupils immediately following pupillary constriction (Figs. 3, 4A). The mechanism underlying the biphasic dilation response is not known. However, we postulate that the initial fast rate of dilation may reflect a relaxation of the iris sphincter muscles, while the slower rate may be attributable to dilator contraction that is initially masked by the action of the sphincter muscles. It has been shown that the time course for dilator muscles that have been stimulated via the nerves connected to them is longer, and can persist after the end of the stimulation pulse. 37 Alternatively, the fast and slower dilation rates may be only related to the sphincter muscle. Pilar and colleagues 37 also show that chicken iris muscles can exhibit both skeletal (fast twitch) and smooth muscle (tetanic contractions) properties; the fast and slow dilation responses may reflect the cessation of these sphincter muscle behaviors. Additional studies are necessary to clearly define the physiological mechanisms underlying the iridial muscles and pupillary behaviors, as observed in our experiments. 
Conclusion
In summary, we demonstrated for the first time that high speed UHR-OCT is able to measure the pupillary responses to visual stimuli in living animals with very high spatial and temporal resolution. Although the current system design is limited only to ipsilateral measurements, future redesign of the imaging system could allow for recording of contralateral pupillary dynamics. By utilizing the UHR-OCT system in a set of animal studies, we found that red light stimuli induced the strongest pupillary contraction, that pupillary dilation following contractions was biphasic, and that magnitude of the pupil constriction was affected negatively by isoflurane anesthesia. Further research needs to be undertaken to determine the physiological mechanisms driving this pupillary behavior. 
Supplementary Materials
Acknowledgments
The authors thank Harmen van der Heide and Krumomir Dworski for assistance with the mechanical and electronic design of the fOCT+ERG system, and Nancy Gibson for assistance with the animal handling. 
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Footnotes
 Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Foundation for Innovation.
Footnotes
 Disclosure: A.A. Moayed, None; V. Choh, None; S. Hariri, None; C. Liu, None; A. Wong, None; K. Bizheva, None
Figure 1. 
 
Schematic of the combined functional UHR-OCT and ERG system with modified imaging probe for imaging the pupil dynamics in chickens: CL, collimating lens; DC, dispersion compensation unit; FC, fiberoptic coupler; M, mirror; NDF, neutral-density filter; PC, polarization controllers; and TS, translation stage. The spectrometer consists of a CL, a volumetric diffraction grating (DG), a focusing lens (FL), and a high-speed InGaAs camera (photodiode array, PDA). The stimulator consists of four color LEDs (white [W], green [G], red [R], and blue [B]), achromat lenses (L1, L2, L3, and L4) and a pellicle (P).
Figure 1. 
 
Schematic of the combined functional UHR-OCT and ERG system with modified imaging probe for imaging the pupil dynamics in chickens: CL, collimating lens; DC, dispersion compensation unit; FC, fiberoptic coupler; M, mirror; NDF, neutral-density filter; PC, polarization controllers; and TS, translation stage. The spectrometer consists of a CL, a volumetric diffraction grating (DG), a focusing lens (FL), and a high-speed InGaAs camera (photodiode array, PDA). The stimulator consists of four color LEDs (white [W], green [G], red [R], and blue [B]), achromat lenses (L1, L2, L3, and L4) and a pellicle (P).
Figure 2. 
 
A representative cross-sectional UHR-OCT image of the chicken iris and pupil. (A) The gray arrow marks the reflective surface of the eye lens, while the black arrow marks the imaging artefact generated by the strong back-reflection of the imaging beam off the corneal apex. (B) The same tomogram with a gray arrow pointing at the pupil edges as determined automatically by the novel pupil edge detection algorithm. (C) A movie of the pupil dynamics.
Figure 2. 
 
A representative cross-sectional UHR-OCT image of the chicken iris and pupil. (A) The gray arrow marks the reflective surface of the eye lens, while the black arrow marks the imaging artefact generated by the strong back-reflection of the imaging beam off the corneal apex. (B) The same tomogram with a gray arrow pointing at the pupil edges as determined automatically by the novel pupil edge detection algorithm. (C) A movie of the pupil dynamics.
Figure 3. 
 
Mean and SEM of the fractional pupil size changes as a function of time for multiple recordings obtained from one animal (A) blue, (B) green, (C) red light stimuli of the same duration. (DF) Comparison of mean pupil dynamics recordings acquired from different animals for the three colors of the visual stimulus. (GI) Mean and SEM of the fractional pupil size changes as a function of time for all chickens for the three colors of the visual stimulus.
Figure 3. 
 
Mean and SEM of the fractional pupil size changes as a function of time for multiple recordings obtained from one animal (A) blue, (B) green, (C) red light stimuli of the same duration. (DF) Comparison of mean pupil dynamics recordings acquired from different animals for the three colors of the visual stimulus. (GI) Mean and SEM of the fractional pupil size changes as a function of time for all chickens for the three colors of the visual stimulus.
Figure 4. 
 
Statistical analysis of the pupillary dynamics data. (A) The average recordings of the fractional pupil size change as a function of time for all chickens for blue, green, and red color flashes and their fitted functions. (B) Slope of the first phase of dilation (S1) for all three colors. (C) Statistical summary from ∼90 pupil dynamics recordings from 10 chickens for the maximum pupil constriction and its latency with respect to the onset of the visual stimulus for the blue, green, and red color stimuli.
Figure 4. 
 
Statistical analysis of the pupillary dynamics data. (A) The average recordings of the fractional pupil size change as a function of time for all chickens for blue, green, and red color flashes and their fitted functions. (B) Slope of the first phase of dilation (S1) for all three colors. (C) Statistical summary from ∼90 pupil dynamics recordings from 10 chickens for the maximum pupil constriction and its latency with respect to the onset of the visual stimulus for the blue, green, and red color stimuli.
Figure 5. 
 
The effect of isoflurane anesthesia on the pupil constriction and subsequent dilation measured at different time points between 5 and 40 minutes relative to the onset of the anesthesia for blue (A), green (B) and red (C) color of the visual stimulus.
Figure 5. 
 
The effect of isoflurane anesthesia on the pupil constriction and subsequent dilation measured at different time points between 5 and 40 minutes relative to the onset of the anesthesia for blue (A), green (B) and red (C) color of the visual stimulus.
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