Fig. 1. Schematic diagram of two-photon excited fluorescence confocal microscopy system. The abbreviations in the figure are as follows: G&R Scanner is a Galvo-resonance scanner, LP is a long-pass filter, and PMT is a photomultiplier tube.

Fig. 2. Temperature as a function of time t and distance x: (a) When the distance is constant, the temperature decays with increasing time; (b) when the time is constant, the temperature decays with increasing distance. In this model, a rectangular pulse of 520 W·cm^–2 with an interval of 0.5 ms is selected as the excitation light source.

Fig. 3. (a) Visible near-infrared absorption spectra and (b) TEM images of GNRs and GNRs-SA, scale bar: 100 nm. the absorption peaks of GNRs\GNRs-SA in the near infrared region were at 820 nm and 800 nm, respectively.

Fig. 4. Photoactivation SH-SY5Y cells imaging of only Fluo-4 labeled and labeled with Fluo-4, AM, Con A and GNRs-SA. (a) The fluorescence images of Fluo-4, AM and GNRs-SA labeled SH-SY5Y cells at different times. (b) the fluorescence images of Fluo-4, AM labeled SH-SY5Y cells at different times. (c) the changing of relative fluorescence intensity of Fluo-4, AM, Con A and GNRs-SA labeled SH-SY5Y cells with time. the numbers “1, 2, 3, 4” correspond to the time points (25 s, 54 s, 99 s, 116 s) of the image in (a), and the four curves correspond to the cells circled in different colors in (a). (d) The changing of relative fluorescence intensity of Fluo-4, AM labeled SH-SY5Y cells with time. the numbers “1, 2, 3, 4” correspond to the time points (1 s, 32 s, 127 s, 187 s) of the image in (b). four curves correspond to cells circled in different colors in (b).

Fig. 5. For Fluo-4, AM labeled and Fluo-4, AM, Con A and GNRs-SA labeled SH-SY5Y cells at the same time, the average photoactivation (cell number n = 300) minimum excitation time and the changes in relative fluorescence intensity caused by photoactivation.

Fig. 6. Verify the changing of the source of Ca^{2 +} during photoactivation. Among them, FITC indicates the use of a 500—550 nm band-pass filter to collect fluorescence, BF indicates transmission imaging, and Merge indicates an overlay of two channels. (a) Fluorescence and TD image of SH-SY5Y cells with Fluo-4, AM staining (scale bar: 50 μm). (b) Two-photon fluorescence images of SH-SY5Y cells labeled with Fluo-4, AM at different times. (c) Fluorescence and TD images of SH-SY5Y cells with Fluo-4, AM staining after adding 2 µmol/L Con A, GNRs-SA (scale bar: 50 μm). (d) Time-series two-photon fluorescence images of SH-SY5Y cells labeled with Fluo-4, AM staining after adding 2 µmol/L Con A, GNRs-SA. (e) The change curves of the ΔF/F of the red virtual circle areas in panel (b) and (d) after adding 200 µmol/L BPATA and 200 µmol/L CaCl₂.