Epi fluorescence eclipse ti microscope

Cells were plated in a glass-bottom petri dish, 35 mm, for 24 h (No. 1.5, MatTeK Corporation, USA) and transiently transfected with the fluorescence resonance energy transfer (FRET)–based ER-targeted cameleon (D1ER) (Palmer et al., 2004 (link)). Cells were incubated in Ca²⁺ imaging solution (in mM: 145 NaCl, 5.4 KCl, 0.5 MgCl₂, 1.2 CaCl₂, 5 HEPES, 5.5 glucose, 0.3 NaH₂PO₄, pH 7.4) and imaged by an Epi-fluorescence Eclipse Ti microscope with a Plan-Fluor 40×/1.3 Oil objective (Nikon, Japan). Emission ratio imaging of the cameleon was accomplished by excitation wavelengths of 425 nm with a dichroic mirror at 515 nm and two emission filters [475 nm for enhanced cyan fluorescent protein (ECFP) and 535 nm for citrine-yellow fluorescent protein (YFP)] (Chroma Technology Corporation, USA). Changes in ER calcium were expressed as the FRET-to-CFP emission ratio. Exposure times were typically 100–200 ms, and images were collected every 10–20 s. Images were analyzed using MetaFluor software (Universal Imaging Corporation, USA). Fluorescence images were background-corrected, and cells with similar cameleon expression were analyzed.

Cells were plated in a glass-bottom petri dish, 35 mm, for 24 h (No. 1.5, MatTeK Corporation, USA). Cells were incubated in calcium imaging solution (in mM: 145 NaCl, 5.4 KCl, 0.5 MgCl₂, 1.2 CaCl₂, 5 HEPES, 5.5 glucose, 0.3 NaH₂PO₄, pH 7.4) containing 5 μM Fura-2 AM (F1221, Thermo Fisher Scientific, USA) or 5 μM Rhod-2 AM (R1245MP, Thermo Fisher Scientific, USA), supplied with 0.02% Pluronic F-127 (P3000MP, Thermo Fisher Scientific, USA) to help disperse AM ester for 45 min at 37°C, respectively. Cells were illuminated at alternating excitation wavelengths of 340 and 380 nm for Fura-2 or a monochromatic excitation wavelength of 540 nm for Rhod-2 in an Epi-fluorescence Eclipse Ti microscope with a Plan-Fluor 40×/1.3 Oil objective (Nikon, Japan). The emitted fluorescence was recorded at 510 nm for Fura-2 or 575 nm for Rhod-2 with an Andor Zyla scientific complementary metal oxide semiconductor (sCMOS) camera (Oxford Instruments, UK). Exposure time was typically 100–200 ms, and images were collected every 10–20 s. Images were analyzed using MetaFluor software (Universal Imaging Corporation, USA). Fluorescence images were background-corrected, and cells with similar Fura-2 or Rhod-2 fluorescence intensity were analyzed. Nuclear signal was excluded when quantifying the Rhod-2 fluorescence signals.