Spinning-Disk Confocal Imaging of Calcein-Stained Crystals

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).