Aquacosmos ratio system

Isolated taste cells were incubated in Tyrode’s solution containing 5 µM Fura-2 acetoxymethyl ester at 37 °C for 30 min. Ca²⁺ imaging was carried out using the AQUACOSMOS/RATIO system (Hamamatsu Photonics, Hamamatsu, JAPAN). The Fura-2 fluorescence ratio excited at 340 and 380 nm was measured. Since Type III taste cells express voltage-dependent Ca²⁺ channels^{1 (link)}, Tyrode’s solution (high-K⁺) with a high concentration of K⁺ was used to identify Type III taste cells. The high-K⁺ Tyrode’s solution contained 90 mM NaCl, 50 mM KCl, 2 mM CaCl₂·2H₂O, 1 mM MgCl₂·6H₂O, 5 mM NaHCO₃, 10 mM HEPES, 10 mM D(+)-glucose, and 10 mM Na pyruvate (pH 7.3 adjusted with NaOH).

INS-1D cells were loaded with 4 μM Fura PE-3/AM for 2.5 h at 37 °C in a chamber. Then the cells were continuously superfused with HK solution at a flow rate of 1 mL/min and observed by an inverted fluorescence microscope (IX71, Olympus, Tokyo, Japan). [Ca²⁺]_i was measured by dual-wavelength fluorometry (alternating excitation, 340 and 380 nm; emission, 510 nm) with an Aquacosmos/Ratio system (Hamamatsu Photonics, Hamamatsu, Japan). The 340/380 ratio fluorescence images indicating relative [Ca²⁺]_i were captured every 10 s. Glucose (8.3 mM) and/or DANA (10 μM) were applied in the superfusing HK solution. INS-1D cells showing increases in [Ca²⁺]_i by responding to 0.3 mM tolbutamide were identified as healthy β-cells^{42 (link)}.

In this experiment, we mainly used a spinning-disk confocal imaging system equipped with an Eclipse Ti-U inverted epifluorescence microscope (Nikon, Tokyo, Japan), hand-made reflection light, CSU-X1 laser-scanning unit (Yokogawa, Tokyo, Japan), and ImagEM C9100-13 electron-multiplying charge-couple device (EM-CCD) camera (Hamamatsu Photonics, Hamamatsu, Japan). The system was operated by a Hamamatsu Photonics AQUACOSMOS/RATIO system. During time-lapse confocal imaging, the exposure time was set to 200 msec, and calcein signals were recorded at 10-min intervals using a 488 nm excitation light and a 505–540 nm bandpass filter. We used another confocal system (A+confocal microscope system; Nikon) that was equipped with a high-resolution galvano scanner and operated by NIS Elements software (Nikon) to visualize crystallization at the cellular level. Calcein was excited at 480 nm and fluorescence was detected at 510–530 nm. Each individual specimen was placed on a glass-based dish and filled with 2 mL of FSW-calcein (100 µM) at room temperature (approximately 26 °C). To set the Z=0 µm on the surface of the glass substrate, we marked the crystals on the glass cover slip [8] (link).