Volumetric fluorescence lifetime images of a Tg (kdrl:GFP;pod:nfsB-mCherry) zebrafish embryo²⁵ at 72 hpf.

(a) Lateral projection of false color fluorescence intensity with GFP in green (488-green channel) and mCherry in red (561-red channel). (b,c) Lateral projection image of false color GFP lifetime and mCherry lifetime. Lifetime was rendered as color according to the color index on the right and intensity was rendered as brightness. (d–f) Dorsal projections of fluorescence intensity, GFP lifetime and mCherry lifetime. (g–i) Cross section fluorescence intensity, GFP lifetime and mCherry lifetime images of the zebrafish head at the upper dotted line marked in (a). (j–l) Cross section fluorescence intensity, GFP lifetime and mCherry lifetime images of the zebrafish kidney at the lower dotted line marked in (a). The color index of all fluorescence lifetime images is shown to the right. Scale bar, 500 μm.

Dorsal, lateral and anterior projections of a 33 hpf Tg(enpep:rtTA; P_Tight:CD2V) zebrafish embryo expressing CD2V sensor in kidney tubules, before and after being treated with 3 mM EGTA, 100 μM BAPTA-AM and 10 μM ionomycin for 2 hours.

(a,c) False color fluorescence intensity projections before and after the treatment, with CFP in blue (405-blue channel) and Venus in green (488-green channel). (b,d) CFP lifetime varies along the kidney tubule. After the treatment, CFP lifetime increased, on average, by 0.1 ns over the entire kidney tubule. (e) Average CFP lifetimes of 6 segments of kidney tubules divided equally, before and after the Ca²⁺ treatment. Despite significant variations in average CFP lifetimes between segments, CFP lifetimes in all segments increased after the treatment. Scale bar, 500 μm.

Dorsal, lateral and anterior projections of a 48 hpf Tg(enpep:rtTA; P_Tight:GEpacmC) zebrafish embryo expressing GEpacmC sensor in kidney tubules and neurons, before and after being treated with 100 μM forskolin and 400 μM IBMX for 2 hours.

(a,c) False color fluorescence intensity projections before and after the treatment, with GFP in green (488-green channel) and mCherry in red (561-red channel). (b,d) GFP lifetime before and after treatment, showing tissue specific variation, which may indicate variations in cAMP level. After the treatment, GFP lifetime increased by 0.2 ns evenly over all tissue types. (e) Average GFP lifetimes in 6 segments of kidney tubules divided equally along the tubule, before and after the cAMP treatment. All segments exhibited GFP lifetime increase after the treatment. Scale bar, 500 μm.

Dorsal, lateral and anterior projections of a 33 hpf Tg(enpep:rtTA; P_Tight:CD2V;P_Tight:GEpacmC) zebrafish embryo expressing both CD2V sensor and GEpacmC sensor, before and after being treated with 3 mM EGTA, 100 μM BAPTA-AM and 10 μM ionomycin for 2 hours to decrease cellular Ca²⁺ level.

(a,e) False color fluorescence intensity projections before and after treatment, with 405-blue channel in blue, 488-green channel in green and 561-red channel in red. (b,f) 405-blue channel fluorescence lifetime (CFP lifetime) increased by 0.10 ns after treatment. (c,g) 488-green channel fluorescence lifetime (intensity weighted average of Venus and GFP lifetimes) was not affected by treatment. (d,h) Recovered GFP lifetime in GEpacmC sensor by the triple-channel intensity lifetime analysis. GFP lifetime was not changed by the treatment. (i) Average CFP lifetimes in 6 segments of kidney tubules divided equally, before and after the Ca²⁺ treatment. Despite significant variations in average CFP lifetimes between segments, CFP lifetimes in all segments increased after the treatment. (j) Average recovered GFP lifetimes in 6 tubule segments, before and after the Ca²⁺ treatment. GFP lifetime showed pronounced spatial variation. This spatial pattern of GFP lifetimes was not significantly altered by the Ca²⁺ treatment. Scale bar, 500 μm.

Dorsal, lateral and anterior projections of a 48 hpf Tg(enpep:rtTA; P_Tight:CD2V;P_Tight:GEpacmC) zebrafish embryo expressing both CD2V sensor and GEpacmC sensor, before and after being treated with 100 μM forskolin and 400 μM IBMX for 2 hours to increase cellular cAMP level.

(a,e) False color fluorescence intensity projections before and after treatment, with 405-blue channel in blue, 488-green channel in green and 561-red channel in red. (b,f) 405-blue channel fluorescence lifetime (CFP lifetime) remained the same after treatment. (c,g) 488-green channel fluorescence lifetime (intensity weighted average of Venus and GFP lifetimes) was slightly increased by the treatment. (d,h) Recovered GFP lifetime by the triple-channel intensity lifetime analysis. GFP lifetime increased by 0.1 ns after the cAMP treatment. (i) Average CFP lifetimes in 6 segments of the kidney tubule divided equally, before and after the cAMP treatment. CFP lifetimes in different segments showed significant spatial variation, and were not significantly affected by the cAMP treatment. (j) Average recovered GFP lifetimes in 6 tubule segments, before and after the cAMP treatment. Although GFP lifetime had significant segment-to-segment variation, they all exhibited an increase after the cAMP treatment. Scale bar, 500 μm.