Fig. 5: EO-Flex probes evoke minimal tissue responses compared to multimode fibers commonly used in optogenetic experiments.

a, b Optical images showing 20 µm thick coronal brain sections around the multimode fiber (a) and EO-Flex probe (b) implantation sites. Both the multimode fiber (diameter, 250 µm) and EO-Flex probe (diameter, 12 µm) were advanced to an ~1 mm depth into the cortex. The images were taken 6 days after implantation in heterozygous Cx3cr1-GFP mice with labeled microglia (green). The sections were co-stained with anti-NeuN (blue) and anti-GFAP (magenta) antibodies to label neurons and astrocytes, respectively. c, d Higher resolution image of a zoomed-in region of (a, b) where the probe tips were located. e–g Population analysis (n = 16 sections from two animals) showing the impact of the multimode fiber or EO-Flex probe implantation on neuronal cell numbers (e), astrocyte reactivity as measured by GFAP expression level (f), and microglia reactivity as measured by microglial cell number (g) for the 6-day implantation. Cellular responses were quantified and averaged across two 150 µm × 1 mm analysis regions flanking each insertion site. To distinguish surgery from probe-related tissue responses, an additional craniotomy of comparable size was made 0.7 mm lateral to each device implantation site. The same analysis approach was used to quantify cellular responses at this sham surgery site. Two-tailed paired t-tests determined P values. h, i Coronal brain section optical images taken 30 days after multimode fiber (h) and EO-Flex probe (i) implantation. j, k Higher resolution image of a zoomed-in region of (h) and (i) where the probe tips were located. l–n Corresponding population analysis (n = 16 sections from two animals) showing neuronal cell numbers (l), astrocyte reactivity (m), and microglia reactivity (n) for the 30-day implantation. Two-tailed paired t-tests determined P values. The following convention was used to indicate P values: “ns” indicates P > 0.05, “*” indicates 0.01 < P ≤ 0.05, “**” indicates 0.001 < P ≤ 0.01, and “***” indicates 0.0001 < P ≤ 0.001. All bar plots are presented as mean ± s.e.m.