Revolution 600

Mammalian live cell imaging was performed as described earlier [28 (link)]. Briefly, transient transfection of the HeLa Kyoto cells was performed in a 24-well format using lipofectamine reagent according to the manufacturer’s protocol. Cells were cultured using DMEM medium supplemented with 10% FBS, glutamine, 50 U/mL penicillin, and 50 U/mL streptomycin, at 37 °C and 5% CO₂. HeLa cell cultures were imaged 24–72 h after the transient transfection using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor Technology, Belfast, UK) equipped with an inverted Nikon Eclipse Ti-E/B microscope (Nikon Instruments, Tokyo, Japan), a 75 W mercury–xenon lamp (Hamamatsu, Hamamatsu, Japan), a 60× oil immersion objective NA 1.4 (Nikon, Tokyo, Japan), a 16-bit Neo sCMOS camera (Andor Technology, Belfast, UK), a laser module Revolution 600 (Andor Technology, Belfast, UK), and a spinning-disk module Yokogawa CSU-W1 (Andor Technology, Belfast, UK). The blue, green, and red fluorescence were acquired using the 405, 488, and 561 nm lasers, a confocal dichroic mirror 405/488/561/640, and filter wheel emission filters 447/60, 525/50, and 617/73, respectively. During imaging, the cells were incubated at 37 °C and 5% CO₂ using a cage incubator (Okolab, Naples, Italy).

Transient transfection of HeLa Kyoto cells and imaging of mammalian cells were performed as described in the Supplementary Methods. The fluorescence of GECIs in the transfected mammalian cells was acquired using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor Technology, UK) equipped with an inverted Nikon Eclipse Ti-E/B microscope (Nikon Instruments, Japan), a 75 W mercury–xenon lamp (Hamamatsu, Japan), a 60× oil immersion objective NA 1.4 (Nikon, Japan), a 16-bit Neo sCMOS camera (Andor Technology, UK), laser module Revolution 600 (Andor Technology, UK), and spinning-disk module Yokogawa CSU-W1 (Andor Technology, UK). The green fluorescence intensity of the developed green GECIs and the red fluorescence intensity of the control R-GECO1 red GECI expressed in mammalian cells were acquired using the 488 or 561 nm lasers, confocal dichroic mirror 405/488/561/640 and filter wheel, 525/50 or 617/73 emission filters, respectively. The region of interest (ROI) was chosen in the cytosol of the cell and the value of fluorescence intensity was measured using the Andor iQ3.1 software (Build Number: 7.0.0.74, Belfast, UK). The background values were subtracted for each ROI.

HeLa Kyoto cell cultures were imaged 24–48 h after transfection using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor Technology, Belfast, UK) equipped with an inverted Nikon Eclipse Ti-E/B microscope (Nikon Instruments, Melville, NY, USA), a 75 W mercury-xenon lamp (Hamamatsu, Iwata City, Japan), a 60× oil immersion objective NA 1.4 (Nikon Instruments, Melville, NY, USA), a 16-bit Neo sCMOS camera (Andor Technology, Belfast, UK), laser module Revolution 600 (Andor Technology, Belfast, UK), spinning-disk module Yokogawa CSU-W1 (Andor Technology, Belfast, UK), and a cage incubator (Okolab, Pozzuoli, Italy). The green and red fluorescence were acquired using 80% of the 488 nm (17.3 µW/cm² before objective lens) and 80% of 561 nm (62.3 µW/cm² before objective lens) laser powers, confocal dichroic mirror 405/488/561/640 and filter wheel emission filters 525/50 and 617/73, respectively.
For time-lapse imaging experiments with varying Ca²⁺ concentration, 2.5 μM ionomycin was added to cells for imaging calcium indicators in the Ca²⁺-saturated state.