(a), Physiochemical properties of the synthesized upconversion nanoparticles. Upper panel, schematic illustration of the core/shell structure and energy transfer (ET) among lanthanide ions in the NaYF4: Yb, Tm@NaYF4 upconversion nanoparticles (UCNPs). Lower panel, the emission spectrum of NaYF4: Yb, Tm@NaYF4 (solid red line) upon 980 nm CW laser irradiation (15 mW/mm2) superimposed by the absorbance spectrum of recombinant LOV2 protein (dashed blue line). Inset: the bright blue emission could efficiently lighten the background upon NIR light illumination at 980 nm. (b), NIR light-induced changes in the absorption spectra of purified MBP-LOVSoc at different time interval after mixing with UCNPs-Stv and irradiation with a 980 nm laser (1 min at a power density of 30 mW/mm2). After blue (excited at 470 nm as control, blue circle) or NIR (red triangle) light stimulation, the recovery time course of LOV2 absorbance at 450 nm was plotted in the lower panel. (c), Specific targeting of streptavidin-conjugated UCNPs to engineered ORAI1 channels in the plasma membrane of HeLa cells. Left panel, Schematic showing the interaction between streptavidin-coated upconversion nanoparticles (UCNPs-Stv) and the engineered ORAI1 Ca2+ channel that harbors a streptavidin-binding tag (StrepTag) in the second extracellular loop. The mCh-ORAI1StrepTag protein was able to efficiently recruit and anchor UCNPs-Stv to the plasma membrane of transfected HeLa cells. Right panel, Florescence microscopy imaging showing the accumulation of UCNPs-Stv (green, λex: 980 nm, λem: 450–500 nm) on the plasma membrane of cells transfected with mCh-ORAI1-StrepTag. Scale bar, 10 μm. (d), NIR light-triggered Ca2+ influx and NFAT nuclear translocation in HeLa cells coexpressing mCh-ORAI1StrepTag and LOVSoc. Ca2+ influx was monitored by GCaMP6s fluorescence whilst GFP-NFAT translocation was reported by GFP signals. Transfected cells were mixed with UCNPs-Stv (20 μg/μl) and illuminated by a 980-nm CW laser to trigger the Ca2+ influx. The relatively slow onset of Ca2+ influx and NFAT nuclear translocation provided us a time window to quickly capture the green signals without noticeably activating LOVSoc during image acquisition at low excitation energy (<1 μW/mm2). Scale bar, 10 μm. (e), NIR light-induced reversible Ca2+ influx reported by R-GECO1.2. HeLa cells were transfected with an IRES bicistronic pMIG retroviral construct that enabled coexpression of ORAI1StrepTag and mCh-LOVSoc. Transfected cells were mixed with 5 mg UCNPs-Stv and illuminated by a 980-nm laser at 30 mW/mm2 to trigger the Ca2+ influx. Data were shown as mean ± s.e.m. from 12 cells in two independent experiments. (f), Flow cytometry analysis of IFN-γ production in mouse CD4+ T lymphocytes transduced with retroviruses co-expressing mCh-LOVSoc and ORAI1StrepTag. Freshly isolated CD4+ T cells were subjected to in vitro differentiation as described in Figure 2b, incubated with 20 μg/μl UCNPs-Stv and 1 μM PMA, and exposed to overnight NIR light pulse (ON/OFF interval of 30 s, 980 nm, 30 mW/mm2) prior to analysis. (g), NFAT-dependent luciferase expression in vivo triggered by NIR light stimulation. Left, Schematic of experimental setup. HeLa cells were transfected with NFAT-Luc and constructs encoding LOVSoc/ORAI1StrepTag. 48 hr post-transfection, cells were treated with 1 μM PMA, incubated with 10 mg UCNPs-Stv (blue sphere) and implanted to the flanks of mice subcutaneously. The implanted areas were then subjected to NIR light irradiation (red) with a 980 nm CW laser (50 mW/mm2, 30 sec ON, 30 sec OFF for a total of 25 min). Right, Shown were bioluminescence imaging of three representative BALB/c mice, one implanted with HeLa cells expressing NFAT-Luc only (left) and the other two with cells expressing LOVSoc and NFAT-Luc (middle and right). Mice were subjected to NIR light irradiation (left and right) with a 980 nm CW laser. The images were acquired 20 min after receiving a single dose of luciferin (100 μL, 15 mg/ml, s.c.). Luciferase-catalyzed bioluminescence was visualized as false color with the same rainbow scale bar for all acquired images. Red circle, implanted area.