2.1.

Retina Preparation

Animal handling was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Both frog (Rana pipiens) and mouse (Mus musculus) retinas were used to demonstrate the transient phototropic adaptation in the retina.

Frog retinas were selected as primary specimens in this study for several reasons. First, the relatively large size of frog (compared to mouse or other mammalian) photoreceptors allows unambiguous observation of individual photoreceptors. Second, the diameter of frog rods ($\sim 5$ to 8 μm) is much larger than cones ($\sim 1$ to 3 μm),^6,7 and thus, rod and cone photoreceptors can be easily separated based on their cellular diameters. Third, rod and cone numbers are roughly equal in frog retinas,^6,7 and thus, unbiased analysis of rod and cone cells can be readily achieved. Preparation procedures of fresh living whole-mount frog retinas have been reported in previous publications.⁸ Briefly, the frog was euthanized by rapid decapitation and double pithing. After enucleating the intact eye, we hemisected the globe below the equator with fine scissors. The lens and anterior structures were removed before the retina was separated from the retinal pigment epithelium.

Mouse retinas were used to verify the transient phototropic adaptation in mammalians. Five-month-old wild-type mice, which have been maintained for more than 20 generations from an original cross of C57Bl/6J to 129/SvEv, were used in this study. The rd1 allele that segregated in the 129/SvEv stock was removed by genetic crossing and verified as previously described.⁹ Protocols for handling mouse samples were previously reported.¹⁰ Briefly, after the eyeball was enucleated from anesthetized mice, the retina was isolated from the eyeball in Ames media and then transferred to a recording chamber. During the experiment, the sample was continuously superfused with oxygenated bicarbonate-buffered Ames medium, maintained at pH 7.4 and 33°C to 37°C.

2.2.

Experimental Setup

The imaging system was based on a NIR digital microscope that has been previously used for functional imaging of living retinal tissues.¹⁰ A fast digital camera (Neo sCMOS, Andor Technology, Belfast, UK) with pixel size $6\times 6{\mu \text{m}}^2$ was used for retinal imaging. A $20\times$ water immersion objective with 0.5 NA was used for frog experiments. Therefore, the lateral resolution of the system was about 1 μm ($0.61\lambda /\mathrm{NA}$). For mouse experiments, we used a $40\times$ water immersion objective with 0.75 NA which has the lateral resolution of 0.7 μm. The system consisted of two light sources: a NIR (800 to 1000 nm) light for retinal imaging and a visible (450 to 650 nm) light-emitting diode (LED) for retinal stimulation. The duration of the visible flash was 5 ms. Figure 1 illustrates rectangular stimulus patterns with oblique incident angles [Fig. 1(a)] and a circular stimulus pattern with perpendicular incident angle [Fig. 1(b)]. Figures 1(a) and 1(b) were used for the experiments in Figs. 2 and 4, and Fig. 3, respectively. All images of retinas in this article were acquired at $200\text{frames}/\mathrm{s}$.

Fig. 1

Schematic diagram of stimulation patterns. O: objective; and R: retina. Black dash lines indicate the normal axis of retinal surface. Red solid lines indicate the incident directions. Top panels are cross-section view (transverse or $x–z$ plane) and the bottom panels are enface view (axial or $x–y$ plane). (a1) Rectangular stimulus (bottom panel) with 30-deg incident angle with respect to the normal axis of retinal surface (top panel). (a2) Rectangular stimulus (bottom panel) with $-30\text{-}\mathrm{deg}$ incident angle (top panel). (b) Circular stimulus (bottom panel) with 0-deg incident angle (top panel). The retina was placed with the ganglion cell layer facing toward the objective.

Fig. 2

Oblique stimulus-evoked photoreceptor displacements. (a) Near infrared (NIR) image of frog photoreceptor mosaic pattern. Green dashed window illustrates stimulus area. Red rectangle indicates the area shown in Video 1 (QuickTime, 7.7 MB) [URL: http://dx.doi.org/10.1117/1.JBO.18.10.106013.1] which displays a pair of pre- and poststimulus images alternating repeatedly 20 times. Red and green arrows point to rods and cones, respectively. (b) Average displacement of 25 rods and cones which were randomly selected from the stimulus area. The gray shadow indicates the standard deviation. (c) Active ratios of rods and cones at time 200 ms after the onset of the stimulus. Six trials were used. For each trial, 25 rods and cones were randomly selected. Thus, in each trial, the active ratio was calculated as the number of active rods or cones divided by 25. (d) Retinal displacements associated with the 30-deg stimulus [Fig. 1(a1)] at 200 ms. Dynamic changes of retinal displacement maps from $-200$ to 1000 ms are displayed in Video 2 (QuickTime, 6.7 MB) [URL: http://dx.doi.org/10.1117/1.JBO.18.10.106013.2]. (e) Retinal displacements associated with the $-30\text{-}\mathrm{deg}$ stimulus [Fig. 1(a2)] at 200 ms. Each square in (d) and (e) represents a $15\times 15{\mu \text{m}}^2$ area of the retina. Transient displacements within the small square were averaged.

Fig. 3

Photoreceptor displacements and intrinsic optical signal (IOS) responses stimulated by circular stimulus (in transverse plane) with a Gaussian profile (in axial plane). (a) NIR image of frog photoreceptor mosaic. Red rectangle indicated the area shown in Video 3 (QuickTime, 7.9 MB) [URL: http://dx.doi.org/10.1117/1.JBO.18.10.106013.3] which displays a pair of pre- and poststimulus images alternating repeatedly 20 times. (b) Localized retinal displacements associated with circular stimulus. This stimulus had a Gaussian profile in axial plane [Fig. 1(b)]. The same methods as those in Figs. 2(e) and 2(f) were used here to produce the displacement maps. (c) IOS maps. $\mathrm{\Delta }I$ reflected the light intensity change and $I$ was the background light intensity. Zone 1 corresponds to the area within the inner ring. Zone 2 corresponds to the annular area. The stimulus was delivered at time 0. (d) Enlarged view of b3. (e) Enlarged view of c3. Scale bars indicate 100 μm. (f) Dynamic change of the number of active pixels in (b). The pixel with nonzero value was defined as active. (g) Temporal IOS profiles.

2.3.

Dynamic Calculation of Individual Photoreceptor Movements

In order to quantify transient phototropic changes in rod and cone systems, we calculated the displacement of individual rods [Figs. 2(b) and 4(b)] and cones [Fig. 2(b)].The level-set method¹¹ was used to identify the morphological edge of individual rods and cones. Then, the weight centroid was calculated dynamically, allowing accurate registration of the location of individual photoreceptors at nanometer resolution. The same strategy has been used in stochastic optical reconstruction microscopy¹² and photoactivated localization microscopy^13,14 to achieve nanometer resolution to localize individual molecules with photoswitchable fluorescence probes. The three-sigma rule was used to set up a threshold to distinguish silent and active photoreceptors.¹⁰ If the stimulus-evoked photoreceptor shifted above this threshold, then this photoreceptor was defined as active. Otherwise, it was defined as silent. Thus, the active ratio of the rods and cones could be obtained [Fig. 2(c)].

2.4.

Computer Algorithm of Localized Retinal Movements

The activated photoreceptors were displaced due to light stimulations [Figs. 2(c), 2(d), and 3(b)]. In order to quantify the photoreceptor displacements, the normalized cross correlation (NCC) between the poststimulus and prestimulus images was calculated to estimate localized retinal movements. We assume that $I_{ti}(x,y)$ was the image acquired at the time point of $t_i$, where $i=1,2,3,\dots$ was the image index and $(x,y)$ was the pixel position. We took the first image $I_{t1}(x,y)$ as the reference image. For the pixel at the position of $(x_0,y_0)$ from the image $I_{ti}$, there would be a horizontal shift $H_{ti}(x_0,y_0)$ (parallel to the $x$ axis) and a vertical shift $V_{ti}(x_0,y_0)$ (parallel to the $y$ axis) compared to the reference image. At the position of $(x_0,y_0)$ from the image $I_{ti}$, we took a subwindow $W_{ti}$ ($m\times m$ pixels):

Then the correlation coefficient could be calculated between two image matrices defined by Eqs. (1) and (2):

They could be rewritten as a complex number

If

2.5.

IOS Data Processing

In order to test the effect of the phototropic adaptation on the IOS pattern associated with circular stimulus, representative IOS images are illustrated in Fig. 3(c), with a unit of $\mathrm{\Delta }I/I$, where $I$ is the background light intensity and $\mathrm{\Delta }I$ reflects the light intensity change corresponding to retinal stimulation. Basic procedures of IOS data processing have been previously reported.⁸

3.1.

Transient Phototropic Adaptation in Frog Retina Activated by Oblique Stimulation

Figure 2 shows results of phototropic adaptation correlated with oblique light stimulation. Figure 2(a) shows the photoreceptor mosaic pattern. Individual rods [red arrows in Fig. 2(a)] and cones [green arrows in Fig. 2(a)] could be observed. A rectangular stimulus with a 30 deg incident angle [Fig. 1(a1)] was delivered to the retina. Within the stimulation area, photoreceptor displacements were directly observed in NIR images (Video 1).

In order to quantify transient phototropic changes in rod and cone systems, we calculated displacements of individual rods and cones (see Sec. 2). Figure 2(b) shows average displacements of 25 rods and cones randomly selected from the stimulus window. The displacement of rods occurred almost immediately ($<10\mathrm{ms}$) and reached a magnitude peak at $\sim 200\mathrm{ms}$. The magnitude of rod displacement (average: 0.2 μm, with maximum up to 0.6 μm) was significantly larger than that of cone displacement (average: 0.048 μm, with maximum of 0.15 μm). In addition, as shown in Fig. 2(c), the active ratio (see Sec. 2 for definition) of rods was $80\%\pm 4\%$, while $20\%\pm 4\%$ of cones were activated. The observation indicated that the transient phototropic displacement was dominantly observed in rods.

In order to verify directional dependency of the phototropic adaptation, we used template matching with the NCC to compute nonuniform motion in the retina (see Sec. 2).¹⁵ As shown in Video 2 and Fig. 2(d), the stimulus-activated retina shifted to right, i.e., toward the direction of the 30-deg oblique stimulation. In order to confirm the reliability of the phototropic response, the incident angle of the stimulus was switched to $-30\mathrm{deg}$ [Fig. 1(a2)], 5 min after the recording illustrated in Fig. 2(d). Figure 2(e) illustrates the transient movement corresponding to $-30\mathrm{deg}$ stimulus at the same retinal area shown in Fig. 2(d). It was observed that the stimulated retina shifted toward the left [Fig. 2(e)], i.e., in the opposite direction compared to Fig. 2(d). Comparative recording of the 30 and $-30\mathrm{deg}$ stimuli verified that transient photoreceptor movement was tightly dependent on the incident direction of the stimulus light.

3.2.

Transient Phototropic Adaptation in Frog Retina Activated Oblique Stimulation by Circular (Transverse) Stimulation with a Gaussian (Axial) Profile

In addition to the aforementioned oblique stimulation, Fig. 3 shows transient photoreceptor displacements activated by a perpendicular circular stimulus with a Gaussian profile in the axial plane [Fig. 1(b)]. The circular aperture was conjugate to the focal plane of the imaging system. It is well known that cones taper toward the outer segment (OS) and are shorter than rods,⁶ which implies that the OS pattern should have relatively larger extracellular space between photoreceptors when compared to the inner segment (IS) pattern. Therefore, when the tight mosaic pattern of photoreceptors [Fig. 2(a)] was clearly observed, the focal plane was around the photoreceptor IS. Hence, at the more distal position, i.e., the OS, the stimulus light was divergent and became oblique at the edge. However, at the central area, the stimulus light impinged the photoreceptor without directional dependence. Under this condition, only photoreceptors at the periphery of the stimulus pattern underwent displacement, which can be directly visualized in Video 3. Figures 3(b) and 3(d) not only confirmed this phenomenon but also revealed that peripheral photoreceptors shifted toward the center. The number of active pixels [see Eq. (7)] was plotted over time in Fig. 3(f). The rapid displacement occurred almost immediately ($<10\mathrm{ms}$) after the stimulus delivery, reached the magnitude peak at $\sim 200\mathrm{ms}$, and recovered at $\sim 2\mathrm{s}$. It was consistently observed that the stimulus-evoked displacement was rod dominant. Utilizing the same methods employed in Fig. 2(c), rod and cone displacements were quantitatively calculated. Within the annular area in Fig. 3(d), 25 rods and cones were randomly selected for quantitative comparison. $74\%\pm 6\%$ of rods were activated, whereas $24\%\pm 5\%$ of cones were activated at 200 ms after the onset of stimulus (six samples).

3.3.

Correlation of Transient Phototropism and IOS in Frog Retina

We speculated that the transient phototropic changes may partially contribute to stimulus-evoked IOSs, which promised a noninvasive method for spatiotemporal mapping of retinal function.^8,16 IOS images shown in Fig. 3(c) confirmed the effect of IOS enhancement at the edge of the circular stimulus. The edge-enhanced IOS response gradually degraded over time, which was consistent with the change of the photoreceptor displacement [Fig. 3(b)]. In addition, both positive and negative IOS signals, with high magnitude, were observed at the periphery of the stimulus pattern. In contrast, the IOS signal at the stimulus center [Zone 2, Fig. 3(e)] was positive dominant, and the IOS magnitude was weaker than that observed at peripheral area [Zone 1, Fig. 3(e)]. Moreover, time courses of IOS responses were different between Zone 1 and Zone 2 [Fig. 3(g)]. The central IOS curve [black curve in Fig. 3(g)] more resembled the curve of the active pixel number [Fig. 3(f)], which suggested that transient phototropic change primarily contributes to the periphery IOSs.

3.4.

Transient Phototropic Adaptation in Mouse Retina Activated by Oblique Stimulation

In order to verify the transient phototropic changes in mammalians, we have conducted a preliminary study of mouse retinas with oblique stimulation. Unlike large frog photoreceptors (rod: $\sim 5$ to 8 μm, cone: $\sim 1$ to 3 μm), mouse photoreceptors (1 to 2 μm for both rods and cones) are relatively small.^17,18 Although individual mouse photoreceptors [Fig. 4(a)] were not as clear as frog photoreceptors [Figs. 2(a) and 3(a)], we selected representative individual mouse photoreceptors [arrows in Fig. 4(a)], which could be unambiguously isolated from others. Figure 4(b) shows temporal displacements of 10 mouse photoreceptors pointed out in Fig. 4(a). These 10 photoreceptors shifted to the left [arrows in Fig. 4(b)]. Figure 4(c) shows an average magnitude of photoreceptor displacements. As shown in Fig. 4(c), the displacement occurred within 5 ms and reached the peak at 30 ms.

Fig. 4

Stimulus-evoked photoreceptor displacements at the mouse retina. (a) NIR image of mouse photoreceptor mosaic. A $40\times$ objective with 0.75 NA was used. The image size corresponds to a $60\times 60{\mu \text{m}}^2$ area at the retina. The green dashed rectangle indicates the oblique stimulation area. (b) Displacments of 10 photoreceptors over time. The stimulus was delivered at time 0. These 10 photoreceptors were specified by arrows in (a). Green arrows in circles indicate the direction of the displacement at time 30 ms after stimulation. (c) Averaged displacement of 10 photoreceptors. The inset panel shows the same data within the time period from $-0.02$ to 0.1 s.