Table 1.

Specifications for VSD and calcium imaging probes

Modality	Dye	Ex535 1P	Ex780 2P	Em460 (% max)	Em610 (% max)
Ca2+ imaging	Fura-2	0% max at 340 nm	Ca2+-free 8.1 GM	98	0
			Ca2+-bound 0.3 GM
VSD imaging	di-3-ANEPPDHQ	50% max at 510 nm	NA/4 GM	2	95

In VSD imaging, 535 nm light, the center wavelength of the excitation band-pass (BP) filter (F in Fig. 1), effectively excites di-3-ANEPPDHQ in the single photon (1P) excitation process, but not Fura-2. In calcium imaging, two-photon (2P) absorption cross-section at 780 nm for Fura-2 Ca²⁺-free form (8.1 GM) is much larger than the Ca²⁺-bound form (0.3 GM) (Wokosin et al., 2004), causing fluorescence signal to extinguish during neuronal activity (e.g., calcium influx through voltage-gated calcium ion channels). The fluorescence traces for calcium imaging in the subsequent figures were all inverted for visual intuition. For detecting Fura-2 signal, a 460 nm band-pass filter (G in Fig. 1) was chosen based on the peak Fura-2 emission spectra. The 2-photon absorption cross-section for di-3-ANEPPDHQ could not be found, but it was reported to be ∼4 GM for a similar molecule, di-8-ANESPPDHQ (Fisher et al., 2008). This indicates that the 780 nm light could excite di-3-ANEPPDHQ by the 2-photon excitation process, but emission ∼460 nm is very weak, and the majority of emission from VSD dye channel can be blocked by the filters (D and G in Fig. 1).

NA, Not applicable.