Dichroic mirror

The rotation of a polystyrene bead attaching to the γ-subunit was visualized under an inverted microscope (Ti-E; Nikon) equipped with 100× objective lens (Apo TIRF N.A. 1.49; Nikon), a condenser unit (LWD0.52; Nikon), LED (pE-100 660 nm; CoolLed), IR laser (YLM-2-1064-LP; IPG), an optical bench (RS2000TM; Newport), and three CCDs (CCD1 in Fig. 1A, CS8430i; Toshiba Teli, CCD2, Luca; Andor, and high-speed CCD3, LRH20000B; DigiMo). CCD2 and CCD3 were synchronized with TTL signal to identify the moment when optical trapping was turned on with a custom modification in CCD3. Dichroic mirrors, DM1 and DM2 in Fig. 1A, were custom made (Chroma Technology) and purchased (Asahi Spectra), respectively. A highly-stable customized sample-stage (Chukousha) was adjusted three actuators (SGSP-13ACTR; Sigma Koki). The optical system for 3-D tracking system was described previously^{14 (link),36} ; the calibration factor to determine z-position from Δx was set as 1 in all measurements. Most apparatuses except PCs and displays were compartmented in a custom-made thermostatic chamber (Nihon Freezer), and all operations were done from the outside of the chamber. The Measurements were done at 23 ± 0.2 °C. speed of camera was set as 30 and 500 f.p.s. for low and high-load, respectively.

Arabidopsis seedlings were grown vertically in 1/2MS solid medium for 7 d. Samples were then gently mounted on a perfusion chamber and stabilized with cotton wool soaked in 200 μl of imaging buffer (10 mM MES Tris base; 1 mM CaCl₂; 5 mM KCl pH 5.8); an ISMATEC pump was used to administrate stimuli in continuous, setting the flow rate at 3 ml/min; each stimulus was added into the imaging buffer. The seedlings were kept under continuous perfusion. For Cameleon (Krebs and Schumacher 2013 (link)) analysis, the FRET CyanoFP/YellowFP (CFP/YFP) optical block A11400-03 (Emission 1, 483/32 nm for CFP and Emission 2 542/27 nm for the FRET) with a dichroic 510 nm mirror (Hamamatsu Photonics, Shizuoka, Giappone) was used for the simultaneous CFP and FRET acquisitions (cpVenus for YC3.6 and YC4.6). Images were acquired in 5-s intervals with an exposure time of 500 ms. Filters and dichroic mirrors were purchased from Chroma Technology, Olching, Germany. The NIS-Element (Nikon) was used to control the microscope, illuminator, camera, and post-acquisition analyses, as previously reported (Giovanna Loro et al. 2016 (link)). FRET efficiency values were calculated as previously described (Loro et al. 2013 (link)).