Growing Arabidopsis pollen tubes expressing genetically encoded reporters for calcium and pH

Spatiotemporal dynamics of intracellular pH (H+) and calcium (Ca2+) in growing Arabidopsis pollen tubes. Fluorescence time-lapses of pollen tubes expressing ratiometric sensors under the pollen-specific LAT52 promoter, namely CaMeleon (Ca2+) [Damineli et al., 2017; DOI: 10.1093/jxb/erx032] and pHluorin (pH) [Hoffmann et al., 2020; DOI: 10.1038/s41467-020-16253-1], were acquired every 4 seconds with an inverted microscope equipped with a front-illuminated camera (2560 × 2160, pixel size 6.45 μm), a fluorescence illuminator, and a ×63 1.2NA water immersion objective lens. The CaMeleon filters were set as follows: excitation 426-450 nm (CFP) and 505-515 nm (YFP), emission 458-487 nm (CFP) and 520-550 nm (YFP). As for pHluorin, the filter settings were: excitation 318-390 nm (DAPI) and 428-475 nm (FITC), emission 435-448 nm (DAPI) and 523-536 nm (FITC) [from Damineli et al., 2017; Hoffmann et al., 2020].