Zinpyr 1

Full-thickness skin set aside for en face imaging was stained with acriflavine hydrochloride (Sigma-Aldrich, Castle Hill, Australia) 100 µM in water overnight. Skin cryosections were stained with 10 µM Zinpyr-1 (Cayman Chemical Co., Ann Arbor, MI, USA).
For multiphoton microscopy (MPM), the 488 nm argon laser was used to excite Zinpyr-1, while the 800 nm tuneable titanium-sapphire Mai-Tai was used to excite ZnPT. Emission was collected from the descanned line under a filter (520–560 nm) for single-photon excitation and a non-descanned filter (395–420 nm) for two-photon excitation. A 40X water immersion objective was used.
Fluorescence lifetime imaging microscopy (FLIM) took place along the non-descanned line of the Zeiss LSM710 microscope, fitted with two bh GaAsP hybrid detectors HPM100-40 and two TCSPC modules SPC-152 (Becker & Hickl GmbH, Berlin, Germany). The samples were excited at 740 and 800 nm. Emission was collected using a 405/10 and 540/20 nm bandpass filter (Semrock Inc., Rochester, NY, USA). Images were captured over 5 min. For en face imaging, acriflavine-stained skin was imaged from surface, 40 and 90 µm using the same FLIM parameters as for Zinpyr-1.
Zin-pyr-1 cryosections were first imaged by MPM using the tile-scan function to capture an entire follicle. FLIM images were then acquired along the length of the follicle for detection of ZnPT (λ_exc = 740 nm).

Seven-day-old Col-0 and sic1 plants grown on 1/2 MS plates were treated with PBS solution containing 20 mM Zinpyr-1 (Cayman Chemical, Ann Arbor, MI) at room temperature in darkness for 3 hours. Then, the seedlings were washed with PBS solution twice. The stained plants were observed with a confocal laser scanning microscope (Leica TCS SP8) using excitation at 490 nm and emission at 530 nm.
For CoroNa staining, 6-day-old Col-0 and sic1 plants grown on 1/2 MS plates were treated with 5 μM CoroNa water solution in the dark for 20 minutes. The stained plants were washed in water twice and then observed under a confocal laser scanning microscope (Leica TCS SP8) with an excitation wavelength of 492 nm and an emission wavelength of 516 nm.