Fig 1.
Expression of GCaMP6s and ChrimsonR-tdTomato in mouse retinae.
(A-D) Fluorescent fundus images showing (A) GCaMP6s expression in wild-type (WT) mouse retina at age P64, 20 days after injection of AAV2.GCaMP6s, (B) GCaMP6s expression in rd10 mouse retina not treated with ChrimsonR at age P43, 19 days after injection, and (C & D) GCaMP6s expression and ChrimsonR-tdTomato co-expression in rd10 mouse retina at age P93, 49 days after injection of AAV2.GCaMP6s and AAV2.ChrimsonR. Scale bar in D indicates 500 μm. (E-G) Vertical section of an rd10 retina showing Hochst-stainned nuclei (E), expression of GCaMP6s (F) and ChrimsonR-tdTomato (G). Cell bodies co-labeled with GCaMP6s and ChrimsonR are indicated by arrows. Tissue was harvested from a mouse aged P240, 196 days after injection. Scale bar in G indicates 50 μm. (H & I) GCaMP6s fluorescent neurons in a WT mouse at age P50, 26 days after injection, (H) and rd10 mouse treated with ChrimsonR at age P112, 69 days after injection, (I) imaged in vivo using an adaptive optics ophthalmoscope focused at the ganglion cell layer. Scale bar in I indicates 10 μm.
Fig 2.
Action spectra of mouse photoreceptors, GCaMP6s, ChrimsonR, and light sources for FACILE.
Normalized action spectra of mouse photoreceptor opsins, melanopsin, ChrimsonR, GCaMP6s excitation and emission, and measured spectra of light sources: 488 nm GCaMP6s excitation laser, and 365 nm and 620 nm stimulating LEDs.
Fig 3.
Computing neuronal responses from in vivo calcium imaging data.
(A) GCaMP6s fluorescent retinal neurons in a WT mouse retina imaged in vivo using an adaptive optics ophthalmoscope focused at the ganglion cell layer. (B) Color map showing normalized response amplitude to the UV (365 nm) stimulus. (C) Color map of response phase to the 365 nm stimulus. (D) Cell segmentation mask constructed from fluorescence intensity image (B) and response color maps (C & D). Scale bar indicates 10 μm and applies to A-D. (E & F) Response time courses of cells indicated in A-D. Shaded bars indicate presentation of the 365 nm LED at 20 μW. Three consecutive trials are shown, the first at the top. Cell E shows ON responses to UV light. Cell F shows OFF responses to UV light.
Fig 4.
Single cell responses to visual stimulation.
Scatter plot of single cell responses to UV (365 nm, 33.6 mW.cm-2 at the retina) and red (620 nm, 168.1 mW.cm-2 at the retina) flashing LED stimuli. Each data point represents one cell. (A) Recordings from WT mice injected with only GCaMP6s at age P71, 27 days after injection. (B) Recordings from two separate groups of rd10 mice injected with only GCaMP6s. The first group was imaged at age P50, 26 days after injection, and the second group was imaged at age P99, 70 days after injection. (C) Recordings from one group of rd10 mice injected with both GCaMP6s and ChrimsonR at three time points: P70, P84, and P112, which are 26, 40, and 68 days post injection respectively.
Fig 5.
Tracking responses of individual cells over time in rd10 mice with ChrimsonR.
(A & B) Images of GCaMP6s fluorescent neurons in an rd10 mouse eye treated with ChrimsonR imaged at two different time points, P70 (A) and P112 (B), at the same location. Scale bar in B indicates 10 μm. (C) Responses of individual cells (n = 137) to red (620 nm) light stimulation that were measured at both ages P70 and P112. Gray thin lines represent single cells. The thick red line shows the mean and SD. Data were pooled from seven retinal locations, from three animals. (D & E) Histograms of response (F1) phase for cells recorded in the same group of rd10 ChrimsonR treated mice, at ages P70 (D) and P112 (E). The number of cells analyzed at P70 was 250, and at P112 was 487.