Fig. 5: CaBLAM-derived BL signals in astrocytes and neurons follow high-amplitude tail movements in head-embedded larval zebrafish. | Nature Methods

Fig. 5: CaBLAM-derived BL signals in astrocytes and neurons follow high-amplitude tail movements in head-embedded larval zebrafish.

From: CaBLAM: a high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase

Fig. 5

a, Confocal image of a 5 dpf Tg(GLAST) larva showing expression of CaBLAM in astrocytes (green) and transmitted light (gray). Scale bar: 2 mm. b, A representative trace (80 s) of detected photons (black) and movement (blue). Blue triangles correspond to detected high-amplitude movements. Scale bars: 50,000 counts (vertical), 10 s (horizontal). c–e, Summed frames from a 4 s video corresponding to a high-amplitude movement: 200 ms before the movement (c), 400 ms of movement (d) and 3,400 ms after the movement (e). Changes are only detectable during the movement, marked by strong uncoordinated and asymmetric tail flicks. f, A zoomed-in view of photons and movement from c–e, marked by the asterisk in b. Counts begin during the movement and continue for seconds after the movement has ceased. Scale bars: 50,000 counts (vertical), 1 s (horizontal). g, Normalized counts during detected high-amplitude movements (gray, n = 10) over a full experiment. Black is the mean BL response. Dotted lines at t = 0 (vertical) and 0 counts (horizontal), vertical scale is −0.25 to 1.25. Scale bar: 10 s. h–k, Normalized counts during high-amplitude movements for four different genotypes. Dotted lines at t = 0 (vertical) and 0 counts (horizontal), vertical scale is −0.15 to 0.5. Scale bar: 10 s. h, Is(nefma) CaBLAM-expressing fish (black, n = 3) and negative siblings (gray, n = 3) with 1:100 vivazine substrate, 3–4 dpf. i, Tg(GLAST) at 1:1,000, 3 dpf, n = 3. j, Tg(hcrtr2) at 1:100, 3 dpf, n = 3. k, Tg(elavl3) at a lower dose (1:1,000), 3 dpf, n = 3. l, A single 30 s exposure of a 5 dpf Tg(elavl3) fish at 1:100 vivazine. m, Pixelwise s.d. across a stack of 83 30 s exposures. n, A composite of the image in n and an infrared-illuminated image of the fish. o, Maximum intensity projection confocal stack of a 4 dpf Tg(elavl3) larva. p, Maximum projection of a 2-min intensified image of a 4 dpf Tg(elavl3) larva in 1:1,000 vivazine. The vertical white line in the center of the image shows the location of the pixels used for analysis. q, A 2-min kymograph of intensity changes over time in the pixels along the white line in p. White triangles indicate movements, the white arrow shows the elevated intensity that followed a large tail flip. Scale bar: 10 s.

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