Orca er

The intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in individual cells were measured with the fluorescent Ca²⁺ indicator fura-2 by dual excitation using a fluorescent-imaging system controlling illumination and acquisition (Aqua Cosmos, Hamamatsu Photonics, Hamamatsu, Japan) as described previously [57 (link)]. To load fura-2, cells were incubated for 40 min at 37°C with 10 μM fura-2 AM (Molecular Probes) in HEPES-buffered solution (in mM: 134 NaCl, 6 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 5 glucose, and 10 HEPES, pH 7.4). A coverslip with fura-2-loaded cells was placed in an experimental chamber mounted on the stage of an inverted microscope (Olympus IX71) equipped with an image acquisition and analysis system. Cells were illuminated every 5 s with lights at 340 and 380 nm, and the respective fluorescence signals at 500 nm were detected. The fluorescence emitted was projected onto a charge-coupled device camera (ORCA-ER, Hamamatsu Photonics) and the ratios of fluorescent signals (F₃₄₀/F₃₈₀) for [Ca²⁺]_i were stored on the hard disk of a computer. Cells were continuously superfused with the external solution at a flow rate of ∼ 2 ml/min. The composition of high-KCl solution was (in mM) 80 KCl, 60 NaCl, 1.2 MgCl₂, 2.5 CaCl₂, and 10 HEPES (pH 7.4 with NaOH). All experiments were carried out at room temperature (22–25°C).

A stereoscopic fluorescence microscope (SZX16; Olympus, Tokyo, Japan) was used for acquiring fluorescence. A mercury lamp (USH1030; Olympus, Tokyo, Japan) was employed as light source for fluorescence excitation. The wavelength of excitation light was set at 405 nm by using an optical filter (BP400-410; Thorlab, Newton, NJ, USA), and the excitation light illuminated with the energy density of 1 mW/cm² in all experiments. Fluorescence from the sample was corrected with an objective lens (×0.5; Olympus, Tokyo, Japan). The total observation magnification was set at ranging from ×0.35 to ×5.75. The fluorescence was obtained with a monochrome charge coupled device (CCD) camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan) with the exposure time of 1 s via the optimized fluorescence filters with the center wavelength of 536 nm (FF01-536/40–25; Semrock, IDEX, Lake Forest, IL, USA), 635 nm (FF01-635/18–25; Semrock, IDEX, Lake Forest, IL, USA), and 736 nm (FF01-736/128–25; Semrock, IDEX, Lake Forest, IL, USA) as described in this article.

Cells expressing GFP-tagged proteins were observed under a total internal reflection fluorescence (TIRF) microscope (based on the IX71 microscope, Olympus, Tokyo, Japan), as previously described [51 (link)]. The cells were wounded with a nanosecond-pulsed laser (FDSS532-Q, CryLas, Berlin, Germany), and the wound diameter was set as 0.5 µm, as previously described [13 (link)]. Time-lapse fluorescence images were acquired at 40–100 ms exposure and 130–500 ms intervals using a cooled CCD camera (Orca ER, Hamamatsu Photonics, Shizuoka, Japan). The time courses of the fluorescence intensities within the circle (3 µm in diameter), including the wound site, were examined using ImageJ software (http://rsbweb.nih.gov/ij, accessed on 1 September 2022). The fluorescence intensities were normalized by setting the value before wounding to 1 after subtracting the background.

To view bacteria on the root surfaces, seedlings were examined with a Nikon Eclipse TE2000-U microscope equipped with a 60× Plan Apo oil objective lens, and pictures were taken with a Nikon D7000 digital camera (real-time movie) or with a Hamamatsu digital camera (model ORCA-ER; for fluorescence detection). Figure 1 presents frames from Movie S1 in the supplemental material that were selected with the iMovie software. The fluorescence signal was detected using a CFP/yellow fluorescent protein (YFP) dual-band filter set (catalog no. 52019; Chroma). All images were taken at the same exposure time, processed identically for compared image sets, and prepared for presentation using MetaMorph and Photoshop software. Each image is representative of at least 12 root colonization assays performed in three independent experiments.