Figure 1. Panel A shows time-lapse... | Rockefeller University Press

Figure 1.

Mercury lamp illumination modulates Ca2+signaling dynamics in astrocytes. (A and B) Astrocytes were transfected with GCaMP6f. Time-lapse images show Ca2+ events under resting conditions (A’ and B’) and following 4-s mercury lamp illumination (7.3–9 mW, 38 GFP filter set, excitation BP 450/50, emission BP 510/50). Local Ca2+ spikes (A” and B”) occur in overlapping regions. Scale bars: 10 μm (whole cell); 10 μm (zoom in). Related to Videos 1 and 2. (C–E) Quantification of local Ca2+ spikes: approximately twofold increase in total Ca2+ events under Local illumination compared with baseline (C), ∼80% of events arising from preexisting hotspots (D), and ∼20% from new sites (E). Error bars represent the mean ± SEM, n = 5 independent experiments. (F) Representative snapshots of Ca2+ signals (green) in the same astrocyte at rest, during local Ca2+ spikes, and Global Ca2+ waves, alongside the z-axis projection (gray) of all Ca2+ events over the same time window (4 min). (G) Total number of Ca2+ events per cell within a 4-min recording window, triggered via mCherry filter (28–30 mW, 31 mCherry filter set, excitation BP 565/30, emission BP 620/60) illumination. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 10 independent experiments. (H) Total number of Ca2+ events per cell within a 4-min recording window under 488-nm laser excitation at 2.1% (1×) or 4.2% (2×) power. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 10 independent experiments. Refer to the image caption for details.

Panel A shows time-lapse images of astrocytes transfected with GCaMP6f, illustrating calcium events under resting conditions and following local illumination. The images highlight local calcium spikes in distal processes. Panel B presents additional time-lapse images under resting and global illumination conditions, showing calcium events. Panel C is a scatter plot quantifying the fold change in total calcium events, indicating a 2-fold increase under local illumination compared to baseline. Panel D is a scatter plot showing that 80 percent of events arise from pre-existing hotspots. Panel E is a scatter plot indicating that 20 percent of events originate from new sites. Panel F includes representative snapshots of calcium signals in the same astrocyte at rest, during local calcium spikes, and global calcium waves, alongside Z-axis projections of all calcium events over a 4-minute window. Panel G is a paired line graph showing the total number of calcium events per cell within a 4-minute recording window triggered via mCherry filter illumination. Panel H is a paired line graph showing the total number of calcium events per cell within a 4-minute recording window under 488 nm laser excitation at different power levels.

Mercury lamp illumination modulates Ca ²⁺ signaling dynamics in astrocytes. (A and B) Astrocytes were transfected with GCaMP6f. Time-lapse images show Ca²⁺ events under resting conditions (A’ and B’) and following 4-s mercury lamp illumination (7.3–9 mW, 38 GFP filter set, excitation BP 450/50, emission BP 510/50). Local Ca²⁺ spikes (A” and B”) occur in overlapping regions. Scale bars: 10 μm (whole cell); 10 μm (zoom in). Related to Videos 1 and 2. (C–E) Quantification of local Ca²⁺ spikes: approximately twofold increase in total Ca²⁺ events under Local illumination compared with baseline (C), ∼80% of events arising from preexisting hotspots (D), and ∼20% from new sites (E). Error bars represent the mean ± SEM, n = 5 independent experiments. (F) Representative snapshots of Ca²⁺ signals (green) in the same astrocyte at rest, during local Ca²⁺ spikes, and Global Ca²⁺ waves, alongside the z-axis projection (gray) of all Ca²⁺ events over the same time window (4 min). (G) Total number of Ca²⁺ events per cell within a 4-min recording window, triggered via mCherry filter (28–30 mW, 31 mCherry filter set, excitation BP 565/30, emission BP 620/60) illumination. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 10 independent experiments. (H) Total number of Ca²⁺ events per cell within a 4-min recording window under 488-nm laser excitation at 2.1% (1×) or 4.2% (2×) power. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 10 independent experiments.