Fluorescein isothiocyanate (fitc)

The BODIPY fluorescence properties were investigated by fluorescence microscopy with a Ti-U Eclipse inverted microscope (Nikon, Tokyo, Japan) equipped with DAPI (excitation at 360/40 nm and emission at 460/50 nm), FITC (excitation at 480/30 nm and emission at 535/40 nm), and G2A (excitation at 535/50 nm and emission > 590 nm) fluorescence filters (Nikon). The intensity of the fluorescence in each image was analyzed with Image J software version 1.53c (National Institutes of Health (NIH), Bethesda, MD, USA) [106 (link)] by quantifying the corrected total cell fluorescence (CTCF) = integrated density—(area of cell × background mean fluorescence) for each cell and by normalizing it against the respective non-treated control sample at 0 h [107 (link)]. The CTCFs were quantified for at least five cells per image, acquired in more than three random microscopic fields in triplicate for each cell line. All fluorescence studies were performed after replacing the cell culture medium with non-supplemented medium without phenol red (ThermoFisher Scientific).

OsRIP1 labelling with fluorescein isothiocyanate (FITC) (Sigma-Aldrich) was carried out as described previously (Chen et al., 2021 (link)). After incubation with OsRIP1-FITC at desired timepoints, PSB-D cells or BY-2 cells were washed with corresponding medium and observed under a fluorescence microscope (Nikon, Melville, NY, USA) with a filter set Ex488/Em525.

Microscopy was performed using a Nikon Eclipse Ti microscope (Tokyo, Japan) equipped with an EMCCD camera (Photometrics Inc. Huntington Beach, CA, USA) and a 100 x, NA 1.40, oil-immersion phase-contrast objective lens. An X-Cite 120 LED was the light source. Three band-pass filter cubes (FITC, DsRed, and C-FL CY5, all from Nikon Inc.) were used for spectral separation. In both FISH and protein fluorescence experiments, an exposure time of 20 ms was used for phase-contrast images. In FISH experiments, the DsRed filter and the C-FL CY5 filter were used to detect Quasar 570 (exposure time of 500 ms, with an electro-multiplier gain of 200 x) and Quasar 670 (exposure time of 300 ms, with an electro-multiplier gain of 100 x), respectively. In protein fluorescence experiments, the FITC and the DsRed filter cubes were used to detect GFP (exposure time of 500 ms, no electro-multiplication) and mCherry (exposure time of 300 ms, no electro-multiplication), respectively. The power of the LED light was carefully controlled so that no significant photobleaching was detected. Images were collected by an automated scanning function of the microscope with a built-in Perfect Focus System (PFS) and analyzed using the Nikon NIS-elements software package. On average, 3000 single cells per protein sample and 1000 single cells per FISH sample were collected and analyzed.

An optical IR-LEGO system was used for the cell disruption of non-target pollen. An Eclipse Ti2-E microscope (Nikon) with an IR-LEGO-490 mini/E system (Sigma-Koki, Saitama, Japan) was used. An objective lens (CFI PlanApo λ 20×, NA = 0.75, WD = 1.0; Nikon) was used for IR laser focusing, irradiation of the target cells with the IR laser, and imaging of the samples. When the power of the IR laser source was set to 300 mW, the laser power delivered to the samples in the focal plane was 145 mW, as measured using a power meter (Coherent, Santa Clara, CA, USA). The irradiation exposure time for cell disruption of each pollen was set from 1 to 10 ms. Images were captured using a scientific CMOS camera (ORCA-Fusion BT C15440-20UP; Hamamatsu Photonics, Shizuoka, Japan). Bandpass filters used for autofluorescence of pollen grains included fluorescein isothiocyanate (FITC) for the FDA signal, Cy3 for tdTomato fluorescence, and cyan fluorescent protein (CFP) (Nikon). ImageJ software version 1.53j (https://imagej.nih.gov/ij/index.html) was used to generate and analyse the images.

Using a wide-field microscopy system, ECLIPSE Ni (Nikon, Tokyo, Japan) equipped with a DIC microscope and objectives (Nikon), Plan Apo λ × 10 [numerical aperture (NA) = 0.45], Plan Apo λ × 20 (NA = 0.75), and Plan Apo λ × 40 (NA = 0.95), fluorescence and DIC imaging were obtained. Filter sets of fluorescein isothiocyanate [excitation: 460–500 nm, dichroic mirror (DM): 505 nm, emission: 510–560 nm; Nikon] and TxRed (excitation: 540–580 nm, DM: 595 nm, emission: 600–660 nm; Nikon) were used for calcein and autofluorescence derived from soft tissue, respectively. Tiling fluorescence and DIC imaging were sequentially performed to acquire the entire, high-contrast view of the tissue sections using the Plan Apo λ × 10 objective (NA = 0.45). The frame size of a single scan was 1280 × 1024 pixels, with an 8-bit color depth and a pixel size of 0.64 μm. Image processing was performed using NIS-Elements AR imaging software (Nikon, Tokyo, Japan).