DOI: 10.5061/dryad.h70rxwdtn
title: Data from: Light-sensitive Ca2+ signaling in the mammalian choroid

description: The choroid is the thin, vasculature-filled layer of the eye situated
 between the sclera and the retina, where it serves the metabolic needs of
 the light-sensing photoreceptors in the retina. Illumination of the
 interior surface of the back of the eye (fundus) is a critical regulator
 of subretinal fluid homeostasis, which determines the overall shape of the
 eye, but it is also important for choroidal perfusion. Noted for having
 some of the highest blood flow rates in the body, the choroidal
 vasculature has been reported to lack intrinsic, intravascular
 pressure-induced (myogenic) autoregulatory mechanisms. Here, we ask how
 light directly regulates choroid perfusion and ocular fluid homeostasis,
 testing the hypothesis that light facilitates ocular fluid absorption by
 directly increasing choroid endothelial permeability and decreasing
 choroid perfusion. Utilizing ex vivo pressurized whole-choroid and
 whole-eye preparations from mice expressing cell-specific Ca2+ indicators,
 we found that the choroidal vasculature has two intrinsically
 light-sensitive Ca2+-signaling mechanisms: one increases Ca2+-dependent
 production of nitric oxide in choroidal endothelial cells, the other
 promotes vasoconstriction through Ca2+ elevation in vascular smooth muscle
 cells. In addition, we found that choroidal flow, or pressure, modulates
 endothelial and smooth muscle photosensitivity and trans-retinal
 absorption of fluid into the choroid. These results collectively suggest
 that the choroid vasculature exhibits an inverted form of autoregulatory
 control, where pressure- and light-induced mechanisms work in opposition
 to regulate blood flow and maintain fluid balance in response to changes
 in light and dark, aligning with the metabolic needs of photoreceptors.
description: High-speed, high resolution spinning disk confocal/widefield
 imaging obtained from ex vivo pressurized and unpressurized choroid
 preparations.  Images in TIFF format will be generated using commercial
 software (VisiView), but images and metadata are freely accessible using
 open-source software such as ImageJ. Immunofluorescence/brightfield
 microscopy images generated from pinned-down en face retinal vasculatures.
 Raw z-stack images will be saved in either TIFF or AVI format and can be
 freely transformed within open-source software.
description: # Data from: Light-sensitive Ca2+ signaling in the mammalian choroid
 [https://doi.org/10.5061/dryad.h70rxwdtn](https://doi.org/10.5061/dryad.h70rxwdtn) ## Description of the data and file structure This dataset contains all the processed data published in "Light Sensitive Ca2+ Signaling in the Mammalian Choroid". **Figure1A_processed_data.xlsx:** This file contains averaged calcium fluorescence traces from choriocapillaris endothelial cells expressing GCaMP6f during 405 nm light stimulation. Imaging was performed using 470 nm excitation at 5 Hz. ***Figure1_A1_Data tab:*** Column A: Time (s): Time in seconds. A value of 0 s indicates the onset of 405 nm light stimulation. Column B: High Power Cell 1: Raw fluorescence intensity values for Cell 1 during high-power stimulation. Column C: High Power Cell 2: Raw fluorescence intensity values for Cell 2 during high-power stimulation. Column D: High Power Cell 3: Raw fluorescence intensity values for Cell 3 during high-power stimulation. Column E: Low Power Cell 1: Raw fluorescence intensity values for Cell 1 during low-power stimulation. Column F: Low Power Cell 2: Raw fluorescence intensity values for Cell 2 during low-power stimulation. Column G: Low Power Cell 3:Raw fluorescence intensity values for Cell 3 during low-power stimulation. All fluorescence values are unnormalized raw intensity measurements. ***Figure1_A1_Statistiacal_Summary tab:*** This tab contains descriptive statistics and inferential analyses of raw fluorescence intensity measurements from choriocapillaris endothelial cells expressing GCaMP6f under two 405 nm stimulation conditions (High Power: 6.1 × 1014 photons/cm2/s; Low Power: 2.04 × 1014 photons/cm2/s; n = 154 per group). An unpaired two-tailed t-test showed that mean fluorescence was significantly higher in the High Power condition (t(306) = 10.45, p &lt; 0.0001), with variances also significantly different between groups (F(153,153) = 6.103, p &lt; 0.0001). **Figure1B_processed_data.xlsx:** This file contains peak calcium responses from capillary endothelial cells isolated from choroids of cdh5-GCaMP6f mice following stimulation with 640 nm, 470 nm, or 405 nm light. Figure1_B_Data tab: Column A: Experiment Identifier Column B: ΔF/F~o~ recorded under 640 nm of light  Column C: ΔF/F~o~ recorded under 470 nm of light  Column D: ΔF/F~o~ recorded under 405 nm of light  ***Figure1_B_Statistical_Summary tab: *** This tab contains descriptive statistics and inferential analyses of peak ΔF/F₀ calcium responses from capillary endothelial cells isolated from choroids of cdh5-GCaMP6f mice following stimulation with 640 nm, 470 nm, or 405 nm light. A one-way ANOVA showed a significant difference among stimulation wavelengths (F = 147.5, p &lt; 0.0001; R² = 0.9335), with data derived from 5 choroids from 3 mice per group. **Figure1C_processed_data.xlsx:** This tab contains peak calcium responses evoked by 405 nm light in capillary endothelial cells from choroids of cdh5-GCaMP6f mice under different pharmacological conditions. ***Figure1_C_Data tab:***  Column A:  Experimental Identifier Column B: ΔF/F~o~ recorded under control conditions with 2 mM extracellular Ca2+ Column C: ΔF/F~o~ recorded under conditions with 0 mM extracellular Ca2+ Column D: ΔF/F~o~ recorded under conditions in the presence of PTX Column E: ΔF/F~o~ recorded under conditions in the presence of CPA Column F: ΔF/F~o~ recorded under conditions in the presence of YM-254890 Column G: ΔF/F~o~ recorded under conditions in the presence of Suramin  Column H: ΔF/F~o~ recorded under conditions in the presence of U73122 Abbreviations: PTX = pertussis toxin (Gi protein inhibitor); CPA = Cyclopiazonic acid (SERCA blocker); YM-254890 = Gq protein inhibitor; Suramin = broad-spectrum G protein–coupled receptor (GPCR) inhibitor; and  U73122 = phospholipase C inhibitor. ***Figure1_C_Statistical Summary tab:*** This tab contains inferential statistical analyses comparing peak ΔF/F₀ calcium responses across seven treatment groups (n = 60 total observations). A one-way ANOVA showed a significant difference among group means (F(6,53) = 97.34, p &lt; 0.0001; R² = 0.9168), with additional Brown–Forsythe (F(6,53) = 2.566, p = 0.0295) and Bartlett’s tests (corrected statistic = 31.49, p &lt; 0.0001) indicating significantly different variances among groups. **Figure1D_processed_data.xlsx:** This file contains averaged DAF2-DA fluorescence traces from choriocapillaris endothelial cells during 405 nm light stimulation. ***Figure1_D1_Data tab:*** Each column (CellA–CellK) represents raw fluorescence intensity values (arbitrary units, AU) recorded over time from a single endothelial cell. Each row corresponds to a sequential time point (2 sec intervals) acquired during the imaging session. ***Figure1_D1_Statistical_Summary tab:*** This tab contains descriptive statistics summarizing DAF2-DA fluorescence intensity measurements from choriocapillaris endothelial cells during 405 nm light stimulation. ***Figure1_D2_Data tab:*** Column A: Cell – Unique identifier corresponding to an individual endothelial cell. Column B: Peak ΔF/F₀ recorded under Control conditions. Column C: Peak ΔF/F₀ recorded in the presence of L-NAME. Column D: Peak ΔF/F₀ recorded in the presence of YM-254890. All values represent peak normalized fluorescence changes (ΔF/F~0~; unitless), calculated as (F − F~0~) / F~0~ for each individual cell. Each row corresponds to one cell. ***Figure1_D2_Statistical_Summary tab: ***This tab contains inferential statistical analyses comparing three experimental groups (A–C), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(2,24) = 55.89, p &lt; 0.0001; R² = 0.8232), with Brown–Forsythe (F(2,24) = 17.63, p &lt; 0.0001) and Bartlett’s tests (corrected statistic = 96.54, p &lt; 0.0001) indicating significantly different variances among groups (total n = 27 observations across 3 treatments). **Figure1F_processed_data.xlsx:** This file contains representative calcium fluorescence traces from mural cells (arteriolar smooth muscle cells and pericytes) expressing GCaMP6f in NG2-GCaMP6f mice during 405 nm (violet) light stimulation. ***Figure1_F_Data tab***: Column A: Raw calcium fluorescence intensity values recorded from Pericyte Cell 1. Column B: Raw calcium fluorescence intensity values recorded from Pericyte Cell 2. Column C: Raw calcium fluorescence intensity values recorded from Pericyte Cell 3. Column D: Raw calcium fluorescence intensity values recorded from Pericyte Cell 4. Column E: Raw calcium fluorescence intensity values recorded from Smooth Muscle Cell (SMC) 1. Column F: Raw calcium fluorescence intensity values recorded from Smooth Muscle Cell (SMC) 2. Column G: Raw calcium fluorescence intensity values recorded from Smooth Muscle Cell (SMC) 3. Column H: Raw calcium fluorescence intensity values recorded from Smooth Muscle Cell (SMC) 4. All values represent unnormalized raw fluorescence intensity measurements. Each row corresponds to a sequential time point acquired during imaging. **Figure1G_processed_data.xlsx:** This file contains summary data of changes in GCaMP6f fluorescence in arteriolar smooth muscle cells and changes in ciliary arteriolar diameter in response to 405 nm (violet) light stimulation under different pharmacological conditions. ***Figure1_G1_Data tab:***  Column A: Experiment – Experimental replicate identifier (n = 7 independent experiments). Column B: Control – Peak ΔF/F₀ measured under control conditions. Column C: 0 Ca²⁺ – Peak ΔF/F₀ measured under conditions with 0 mM extracellular Ca²⁺. Column D: YM-254890 – Peak ΔF/F₀ measured in the presence of YM-254890. Column E: Nimodipine – Peak ΔF/F₀ measured in the presence of nimodipine. All values represent peak normalized fluorescence changes (ΔF/F₀; unitless) calculated for each independent experiment. Each row corresponds to one experimental replicate. ***Figure1_G1_Statistical_Summary tab: ***This tab contains inferential statistical analyses comparing four experimental groups (Control, 0 Ca²⁺, YM-254890, and Nimodipine; n = 7 per group, 28 total observations), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(3,24) = 28.11, p &lt; 0.0001; R² = 0.7784), with Brown–Forsythe (F(3,24) = 4.501, p = 0.0121) and Bartlett’s tests (corrected statistic = 28.49, p &lt; 0.0001) indicating significantly different variances among groups. ***Figure1_G2_Data tab:***  Column A: Experiment – Experimental replicate identifier (n = 6 independent experiments). Column B: Dark – Measured response under dark (no light stimulation) conditions. Column C: 400 nm – Measured response following 400 nm light stimulation. Column D: 0 Ca²⁺ – Measured response under conditions with 0 mM extracellular Ca²⁺ during stimulation. Column E: Nimodipine – Measured response in the presence of nimodipine during stimulation. Column F: 9-Phen – Measured response in the presence of 9-Phenanthrol during stimulation. All values represent the recorded experimental response for each independent experiment. Each row corresponds to one experimental replicate. ***Figure1_G2_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing five experimental groups (Dark, 400 nm, 0 Ca^2+^, Nimodipine, and 9-Phen; n = 6 per group, 30 total observations), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(4,25) = 29.31, p &lt; 0.0001; R^2^ = 0.8242), while the Brown–Forsythe test indicated no significant difference in variances among groups (F(4,25) = 2.638, p = 0.0577). **Figure1H_processed_data.xlsx:** This file contains summary data of changes in GCaMP6f fluorescence in choriocapillaris pericytes and changes in capillary vessel diameter in response to 405 nm (violet) light stimulation. ***Figure1_H1_Data tab: ***changes in arteriole vessel diameter in response to 405 nm Column A: Experiment – Experimental replicate identifier (n = 8 independent experiments). Column B: Control – Peak ΔF/F~0~ measured under control conditions. Column C: 0 Ca^2+^ – Peak ΔF/F~0~ measured under conditions with 0 mM extracellular Ca^2+^. Column D: YM-254890 – Peak ΔF/F~0~ measured in the presence of YM-254890. Column E: Nimodipine – Peak ΔF/F~0~ measured in the presence of nimodipine. All values represent peak normalized fluorescence changes (ΔF/F~0~; unitless) calculated as (F − F~0~) / F~0~ for each independent experiment. Each row corresponds to one experimental replicate. ***Figure1_H1_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing four experimental groups (Control, 0 Ca^2+^, YM-254890, and Nimodipine; n = 8 per group, 32 total observations), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(3,28) = 54.6, p &lt; 0.0001; R^2^ = 0.854); the Brown–Forsythe test indicated no significant difference in variances (F(3,28) = 1.720, p = 0.1856), while Bartlett’s test detected significantly different variances among groups (corrected statistic = 16.9, p = 0.0007). ***Figure_H2_Data tab: ***changes in capillary vessel diameter in response to 405 nm Column A: Experiment – Experimental replicate identifier (n = 6 independent experiments). Column B: Dark – Measured response under dark (no light stimulation) conditions. Column C: 400 nm – Measured response following 400 nm light stimulation. Rows 2–7 contain individual experimental replicate values. Rows below summarize descriptive statistics for each condition, including Mean, Standard Deviation (SD), Standard Error of the Mean (SEM), and the lower and upper bounds of the 95% confidence interval (CI) of the mean. **Figure2B_processed_data.xlsx:** This file contains a representative calcium fluorescence trace and summary data from choriocapillaris endothelial cells expressing Cdh5-GCaMP6f during increases in intraluminal pressure from 10 mmHg to 25 mmHg. ***Figure2_B1_Data tab:*** Column A: Raw fluorescence intensity values recorded under control conditions (replicate 1). Column B: Raw fluorescence intensity values recorded under control conditions (replicate 2). Column C: Raw fluorescence intensity values recorded under control conditions (replicate 3). Column D: Raw fluorescence intensity values recorded in the presence of Yoda1 (replicate 1). Column E: Raw fluorescence intensity values recorded in the presence of Yoda1 (replicate 2). Column F: Raw fluorescence intensity values recorded in the presence of Yoda1 (replicate 3). Column G: Raw fluorescence intensity values recorded in the presence of Yoda1 and Ruthenium Red (replicate 1). Column H: Raw fluorescence intensity values recorded in the presence of Yoda1 and Ruthenium Red (replicate 2). Column I: Raw fluorescence intensity values recorded in the presence of Yoda1 and Ruthenium Red (replicate 3). All values represent unnormalized raw fluorescence intensity measurements. Each row corresponds to a sequential time point acquired during imaging. ***Figure2_B1_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing three experimental groups (Control, Yoda1, and Yoda1 + RuR), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(2,54) = 147.4, p &lt; 0.0001; R² = 0.8452), with Brown–Forsythe (F(2,54) = 11.94, p &lt; 0.0001) and Bartlett’s tests (corrected statistic = 32.9, p &lt; 0.0001) indicating significantly different variances among groups. ***Figure2_B2_Data tab:***  Column A: Experimental replicate identifier (n = 9 independent experiments). Column B: Measured response under control conditions. Column C: Measured response in the presence of Yoda1. Column D: Measured response in the presence of Yoda1 and Ruthenium Red (RuR). Rows 2–10 contain individual experimental replicate values. Rows below summarize descriptive statistics for each condition, including Minimum, Maximum, Range, Mean, Standard Deviation (SD), Standard Error of the Mean (SEM), and the lower and upper bounds of the 95% confidence interval (CI) of the mean. ***Figure2_B2_Statistical_Summary tab:***  This tab contains inferential statistical analyses comparing three experimental groups (Control, Yoda1, and Yoda1 + RuR; n = 9 per group, 27 total observations), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(2,24) = 37.09, p &lt; 0.0001; R^2^ = 0.7555), with no significant differences in variances detected by Brown–Forsythe (F(2,24) = 0.1368, p = 0.8729) or Bartlett’s tests (p = 0.8888). Dunnett’s multiple comparisons test showed that Yoda1 responses were significantly increased compared to both Control and Yoda1 + RuR (adjusted p &lt; 0.0001 for both comparisons). **Figure2D_processed_data.xlsx:** This file contains representative calcium fluorescence traces from choriocapillaris endothelial cells expressing Cdh5-GCaMP6f during violet light (405 nm) stimulation under controlled intravascular pressure conditions. ***Figure2_D_Data tab:***  Column A: Raw fluorescence intensity values recorded for experimental replicate 1. Column B: Raw fluorescence intensity values recorded for experimental replicate 2. Column C: Raw fluorescence intensity values recorded for experimental replicate 3. Column D: Raw fluorescence intensity values recorded for experimental replicate 4. Column E: Raw fluorescence intensity values recorded for experimental replicate 5. Column F: Raw fluorescence intensity values recorded for experimental replicate 6. Column G: Raw fluorescence intensity values recorded for experimental replicate 7. All values represent unnormalized raw fluorescence intensity measurements. Each row corresponds to a sequential time point acquired during imaging. **Figure2E_processed_data.xlsx**: This file contains summary data of Cdh5-GCaMP6f fluorescence responses to changes in intravascular pressure (e.g., 0, 25, or 60 mmHg) and violet light stimulation. ***Figure2_E_Data tab:*** Column A: Experimental replicate identifier (n = 4 independent experiments). Column B: Measured response under 0 mmHg pressure conditions. Column C: Measured response under 20 mmHg pressure conditions. Column D: Measured response under 60 mmHg pressure conditions. All values represent the recorded experimental response for each independent experiment under the specified pressure condition. Each row corresponds to one experimental replicate. ***Figure2_E_Statistical_Summary tab:*** This tab contains inferential statistical analysis comparing three pressure conditions (0 mmHg, 20 mmHg, and 60 mmHg; n = 4 per group). A one-way ANOVA demonstrated a significant difference among group means (F = 33.08, p = 0.0001; R^2^ = 0.8921), indicating that pressure level significantly affected the measured response. **Figure2G_processed_data.xlsx:** This file contains summary data of percent changes in ciliary arteriolar diameter in response to violet light stimulation and changes in intravascular pressure. ***Figure2_G_Data tab:***  Column A: Experimental replicate identifier (n = 4 independent experiments). Column B: Measured response under 20:80 mmHg pressure conditions. Column C: Measured response under 20:80 mmHg conditions in the presence of barium (Ba^2+^). Column D: Measured response under 20:80 mmHg conditions in the presence of VL. All values represent the recorded experimental response for each independent experiment under the specified condition. Each row corresponds to one experimental replicate. ***Figure2_G_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing three experimental groups (Data sets A–C; n = 11 total observations) analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(2,8) = 20.29, p = 0.0007; R^2^= 0.8353). The Brown–Forsythe test indicated significantly different variances among groups (F(2,8) = 6.091, p = 0.0247). **Figure3B_processed_data.xlsx:** This file contains summary data of fluorescein fluorescence intensity measured in the retinal pigment epithelium (RPE; top dataset) and choroid tissue (bottom dataset) under different intravascular pressure conditions, violet light stimulation, and pharmacological treatment. ***Figure3_B1_Data tab:***  Column A: Raw fluorescence intensity values recorded under dark conditions at 25 mmHg pressure. Column B: Raw fluorescence intensity values recorded during violet light stimulation at 25 mmHg pressure. Column C: Raw fluorescence intensity values recorded during violet light stimulation in the presence of L-NAME at 25 mmHg pressure. Column D: Raw fluorescence intensity values recorded during violet light stimulation at 60 mmHg pressure. All values represent unnormalized raw fluorescence intensity measurements. Each row corresponds to a sequential time point acquired during imaging. ***Figure3_B1_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing four experimental conditions (Dark 25 mmHg, Violet Light 25 mmHg, Violet Light + L-NAME 25 mmHg, and Violet Light 60 mmHg; total n = 72 observations) analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(3,68) = 51.21, p &lt; 0.0001; R^2^ = 0.6932). Neither the Brown–Forsythe test (F(3,68) = 0.9985, p = 0.399) nor Bartlett’s test (p = 0.1791) indicated significant differences in variances among groups. ***Figure3_B2_Data tab:***  Column A:Measured response under dark conditions at 25 mmHg pressure. Column B:Measured response during violet light stimulation at 25 mmHg pressure. Column C:Measured response during violet light stimulation in the presence of L-NAME at 25 mmHg pressure. Column D:Measured response during violet light stimulation at 60 mmHg pressure. All values represent individual experimental measurements (raw response values). Each row corresponds to one independent observation. ***Figure3_B2_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing four experimental conditions (Dark 25 mmHg, Violet Light 25 mmHg, Violet Light + L-NAME 25 mmHg, and Violet Light 60 mmHg; total n = 112 observations) analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(3,108) = 97.06, p &lt; 0.0001; R^2^ = 0.7294). Both the Brown–Forsythe test (F(3,108) = 8.990, p &lt; 0.0001) and Bartlett’s test (corrected statistic = 36.96, p &lt; 0.0001) indicated significantly different variances among groups. **Figure4D_processed_data.xlsx:** This file contains a representative fluorescence trace and summary data of averaged calcium responses from choriocapillaris endothelial cells expressing Cdh5-GCaMP6f during constant violet light stimulation, in the presence of exogenous 9-cis retinal and following depletion of 9-cis retinal. ***Figure4_D1_Data tab:***  Column A: Raw fluorescence intensity values recorded during violet light (VL) stimulation in the presence of cis-retinal (replicate 1). Column B: Raw fluorescence intensity values recorded during violet light stimulation in the presence of cis-retinal (replicate 2). Column C: Raw fluorescence intensity values recorded during violet light stimulation in the presence of cis-retinal (replicate 3). Column D: Raw fluorescence intensity values recorded during violet light stimulation in the absence of cis-retinal (replicate 1). Column E: Raw fluorescence intensity values recorded during violet light stimulation in the absence of cis-retinal (replicate 2). Column F: Raw fluorescence intensity values recorded during violet light stimulation in the absence of cis-retinal (replicate 3). Column G: Raw fluorescence intensity values recorded during 405 nm stimulation in the absence of cis-retinal (replicate 1). Column H: Raw fluorescence intensity values recorded during 405 nm stimulation in the absence of cis-retinal (replicate 2). Column I: Raw fluorescence intensity values recorded during 405 nm stimulation in the absence of cis-retinal (replicate 3). All values represent unnormalized raw fluorescence intensity measurements. Each row corresponds to a sequential time point acquired during imaging. ***Figure4_D1_Statistical_Summary tab: ***This tab contains descriptive statistics and inferential analyses comparing three experimental conditions: VL (+) cis-retinal, VL (−) cis-retinal, and cis-retinal (−) 405 nm (n = 270 observations per group; 810 total). A one-way ANOVA demonstrated a significant difference among group means (F(2,807) = 1185, p &lt; 0.0001; R² = 0.746). Both the Brown–Forsythe test (F(2,807) = 206.4, p &lt; 0.0001) and Bartlett’s test (corrected statistic = 1590, p &lt; 0.0001) indicated significantly different variances among groups. ***Figure4_D2_Data tab:***  Column A: Measured response under control conditions. Column B: Measured response in the presence of 9-cis retinal. Column C: Measured response in the absence of 9-cis retinal. All values represent individual experimental measurements. Each row corresponds to one independent experimental replicate. ***Figure4_D2_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing three experimental groups (Control, +9-cis, and −9-cis; n = 3 per group, 9 total observations), analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA demonstrated a significant difference among group means (F(2,6) = 46.94, p = 0.0002; R^2^ = 0.9399). The Brown–Forsythe test indicated no significant difference in variances among groups (F(2,6) = 1.279, p = 0.3447). **Supplementary_FigureS1A_processed_data.xlsx:** This file contains representative traces and summary data of averaged Cabryte 630 fluorescence measured in choriocapillaris endothelial cells during 470 nm and 405 nm light stimulation. ***FigureS1_A_Trace_Data tab:*** Columns A–T: ROI 1–ROI 20 – Raw fluorescence intensity values recorded from 20 individual regions of interest (ROIs). Each column represents a single ROI, and each row corresponds to a sequential time point acquired during imaging. All values represent unnormalized raw fluorescence intensity measurements for each ROI. ***FigureS1_A_Data_Summary tab:***  Column A: 470 nm – Measured response during 470 nm light stimulation. Column B: 405 nm – Measured response during 405 nm light stimulation. All values represent individual experimental measurements (e.g., peak ΔF/F₀ or normalized response values, unitless). Each row corresponds to one independent experimental replicate. ***FigureS1_A_Statistical_Summary:*** This tab contains results from an unpaired, two-tailed t-test comparing responses between the 470 nm and 405 nm stimulation conditions. The analysis showed a significant difference between groups (t(4) = 6.705, p = 0.0026), indicating that responses differed significantly at α = 0.05. **Supplementary_FigureS1B_processed_data.xlsx**: This file contains summary data of 405 nm light–mediated changes in Cdh5-GCaMP6f fluorescence in choriocapillaris endothelial cells under different pharmacological conditions. ***FigureS1_B_Data tab:***   Column A: Experimental replicate identifier (n = 4 independent experiments). Column B: Measured response under physiological salt solution (PSS) control conditions. Column C: Measured response in the presence of tetrodotoxin (TTX). Column D: Measured response in the presence of TEMPOL. All values represent individual experimental measurements for each condition. Each row corresponds to one independent experimental replicate. ***FigureS1_B_Statistical_Summary tab:*** This tab contains inferential statistical analyses comparing three experimental groups (PSS, TTX, and TEMPOL; n = 4 per group, 12 total observations) analyzed under the assumption of a normal (Gaussian) distribution. A one-way ANOVA showed no significant difference among group means (F(2,9) = 0.3479, p = 0.7152; R^2^ = 0.07177). Consistent with this, the Brown–Forsythe test (F(2,9) = 0.0493, p = 0.9521) and Bartlett’s test (corrected statistic = 0.383, p = 0.8257) indicated no significant differences in variances among groups. **Supplementary_FigureS2B_processed_data.xlsx:** This file contains representative fluorescence traces and associated image-derived measurements from a Myh11-GCaMP6f-Ai95D mouse choroidal flat-mount preparation showing changes in calcium-dependent fluorescence in response to light stimulation (405 nm and 640 nm) in arterioles and pericytes. ***FigureS2_B_Data tab:*** Columns represent individual regions of interest (ROIs) or peak measurements at specified wavelengths (e.g., 405 nm, 410 nm, 420 nm, etc.), as indicated in the column headers. Each column contains raw fluorescence intensity values corresponding to a specific ROI or wavelength measurement. Each row corresponds to a sequential time point acquired during imaging. All values represent unnormalized raw fluorescence intensity measurements. **Supplementary_FigureS3B_processed_data.xlsx:** This file contains summary data of changes in Fluo-4-AM fluorescence (Arbitrary Units, AU) in primate (Cynomolgus monkey) choriocapillaris endothelial cells following 405 nm (violet) light stimulation under control conditions and in the presence of pharmacological treatment. ***FigureS3_B1_Data tab:*** Columns A–N: Cell 1–Cell 14 – Raw fluorescence intensity values recorded from 14 individual cells. Each column represents a single cell, and each row corresponds to a sequential time point acquired during imaging. All values represent unnormalized raw fluorescence intensity measurements for each individual cell. FigureS3_B1_Statistical_Summary tab: This tab contains descriptive statistics summarizing fluorescence intensity measurements across all analyzed cells. Reported values include the minimum (20,256), maximum (40,704), range (20,448), mean (31,399), standard deviation (7,443), standard error of the mean (544.3), and the lower and upper bounds of the 95% confidence interval of the mean (30,325–32,472). All values represent raw, unnormalized fluorescence intensity ***FigureS3_B2_Data tab:***  Column A: PSS – Measured response under physiological salt solution (PSS) control conditions. Column B: YM-254890 – Measured response in the presence of YM-254890. All values represent individual experimental measurements for each condition. Each row corresponds to one independent experimental observation. ***FigureS3_B2_Statistical_Summary tab: ***This tab contains results from a paired, two-tailed t-test comparing responses under PSS and YM-254890 conditions. The analysis showed a significant difference between paired measurements (t(12) = 10.20, p &lt; 0.0001; n = 13 pairs), indicating that YM-254890 significantly altered the measured response relative to PSS at α = 0.05. **Supplementary_FigureS3C_processed_data.xlsx**: This file contains summary data of changes in arteriole diameter in primate choroidal preparations following 405 nm (violet) light stimulation. ***FigureS3_C_Data tab:*** Column A: Dark – Measured response under dark (no light) conditions. Column B: Light – Measured response under light stimulation conditions. All values represent individual experimental measurements. Each row corresponds to one independent experimental replicate. ***FigureS3_C_Statistical_Summary tab: ***This tab contains results from a paired, two-tailed t-test comparing responses under Dark and Light conditions. The analysis showed a significant difference between paired measurements (t(2) = 8.488, p = 0.0136; n = 3 pairs), indicating that light exposure significantly altered the measured response relative to dark conditions at α = 0.05. **Supplementary_FigureS4B_processed_data.xlsx:** This file contains summary data of pericyte density measured in the choroid and retina from NG2-dsRed mice. ***FigureS4_B_Data tab:*** Column A: Choroid – Measured density of pericytes from choroid samples. Column B: Retina – Measured density of pericytes from retinal samples. All values represent individual experimental measurements. Each row corresponds to one independent biological sample. ***FigureS4_B_Statistical_Summary tab:*** This tab contains results from an unpaired, two-tailed t-test comparing pericyte density between the choroid and retina. The analysis showed a highly significant difference between tissues (t(7) = 69.29, p &lt; 0.0001), indicating that pericyte density differs significantly between the choroid and retina at α = 0.05. **Supplementary_FigureS4C_processed_data.xlsx**: This file contains summary data of pericyte coverage in the choroid and retina from NG2-dsRed mice. ***FigureS4_C_Data tab:***  Column A: Percentage pericyte coverage measured in choriocapillaris vessels from NG2-dsRed mice. Column B: Percentage pericyte coverage measured in retinal capillaries from NG2-dsRed mice. All values represent summary measurements of pericyte coverage (expressed as percent coverage) from individual biological samples. Each row corresponds to one independent sample. ***FigureS4_C_Statistical_Summary tab: ***This tab contains results from an unpaired, two-tailed t-test comparing pericyte coverage between choriocapillaris and retinal capillaries in NG2-dsRed mice. The analysis showed a highly significant difference between groups (t(4) = 34.18, p &lt; 0.0001), indicating that pericyte coverage differs significantly between the choroid and retina at α = 0.05. **Supplementary_FigureS4D_processed_data.xlsx:** This file contains summary data of capillary diameters measured in the choroid and retina from NG2-dsRed mice. ***FigureS4_D_Data tab:*** Column A: Capillary diameter measurements obtained from retinal vessels in NG2-dsRed mice. Column B: Capillary diameter measurements obtained from choriocapillaris vessels in NG2-dsRed mice. All values represent individual capillary diameter measurements. Each row corresponds to one capillary measured. ***FigureS4_D_Statistical_Summary tab: ***This tab contains results from an unpaired, two-tailed t-test comparing capillary diameters between retinal capillaries and choriocapillaris vessels in NG2-dsRed mice. The analysis showed a highly significant difference between groups (t(38) = 6.074, p &lt; 0.0001), indicating that capillary diameters differ significantly between the retina and choroid at α = 0.05. ### Overview of Figures #### Figure 1 **Description:** ***Violet light Stimulate Cell-specific Ca2+ Signals in the Choriocapillaris.*** ##### Variables * Wavelength of light (dark, and 640, 470, and 405 nm light)  * Pharmacological Treatments (Ca2+, CPA, YM254890, Suramin, PTX, U73122, L-NAME, nimodipine, and 9-phenanthrol)  * Cell types (capillary endothelial cells, arterioles smooth muscle cells, and pericytes) #### Figure 2 **Description:** ***: Intraluminal Pressure Regulates Endothelial Opsin Signaling*** ##### Variables * Pharmacological Treatment (Yoda1, Ruthenium red, and Barium) * Intraluminal Pressures (20, 25, 60, 80 mmHg) * Light vs Dark #### Figure 3 **Description:** ***Photomechanical control of subretinal fluid absorption.*** ##### Variables * Examination of RPE and Choroid  * Light vs Dark  * Intraluminal Pressure (25 and 60 mmHg)  * Pharmacological treatments (L-NAME) #### Figure 4 **Description:** ***Expression of OPN3, OPN4, and OPN5 photopigments in the choroid vasculature*** ##### Variables * Reporter mice (OPN3, OPN4, and OPN5) * Pharmacological treatments (exogenous 9-cis and depleated 9-cis)  * Light vs Dark #### Supplementary Figure S1 **Description:** ***Violet light Stimulate Vasculature Ca2+ Signals*** ##### Variables * Model of Ca2+ imaging (Cabryte-630 and cdh5-CCaMP6f)  * Light Treatment (470-405 nm) * Pharmacological Treatment (Tetrodotoxin and TEMPOL) #### Supplementary Figure S2 **Description:** ***Violet light stimulates Ca2+ and Constriction in Choroids Isolated from Myh11-GCaMP6f Transgenic Mice*** ##### Variables * Arteriole vs Pericytes  * Dark vs Light (405 and 640 nm)  #### Supplementary Figure S3 **Description:** ***Intrinsic Light-Sensitive Ca2+ Signaling in the Primate Choriocapillaris Endothelium*** ##### Variables * Dark vs Light (405 nm)  * Pharmacological Treatment (YM-254890) #### Supplementary Figure S4 **Description:** ***Choroid Express Greater Number of Capillary Pericytes*** ##### Variables * Choroid vs Retinal Vascular Beds  ## Code/software None ## Access information Other publicly accessible locations of the data: * None Data was derived from the following sources: * High-speed, high resolution spinning disk confocal/widefield imaging obtained from *ex vivo* pressurized and unpressurized retinal preparations. Images in TIFF format will be generated using commercial software (VisiView) but images and metadata are freely accessible using open source software such as ImageJ.