Plan apochromat dic h

The results were registered with the Leica SP8 confocal microscope using lasers emitting light at wavelengths of 405, 488 and 561 nm with an optimized pinhole, long exposure time (200 kHz) and magnification ×63 (numerical aperture, 1.4). For the Leica confocal microscope Plan Apochromat DIC H an oil immersion lens was used. To minimize bleed-through between fluorescence channels, the low laser power (0.4–5% of maximum power) and single-channel collection were applied.
Living A. thaliana roots were observed using the Nikon A1RSi confocal microscope working with Nikon NIS Elements AR software. Lasers emitting light at wavelengths of 405 and 488 nm with 450/50 nm and 525/50 nm emission filters were used. The pinhole and exposure time were optimized. Plan Apo VC ×20 with numerical aperture 0.75 and Plan Apo VC ×60 with numerical aperture 1.2 (water immersion) objectives were used. To minimize bleed-through between fluorescence channels, the low laser power (0.5–5% of maximum power) and single-channel acquisition were applied. Pinhole sizes for examined channels were matched and the optical section thickness for axial acquisition of defined imaging depths was optimized according to Nyquist criteria as automatically set by Nikon NIS Elements AR software.

In vitro elongated WT and siPhCRT3a pollen tubes were transferred to liquid culture medium containing 400 μM m-maleimidobenzoyl-N-hydroxysuccinimide ester (Merck, Darmstadt, Germany) for 6 min. Next, pollen tubes were permeabilized with an actin-stabilizing buffer (ASB) composed of 100 mM PIPES, 5 mM MgSO₄, 0.5 mM CaCl₂, and 0.05% Triton X-100 (pH 9.0) for 5 min, and fixed with freshly prepared 2% formaldehyde in ASB at room temperature for 30 min. Pollen tubes were washed three times with ASB as above, except at pH 7.0, and supplemented with 10 mM EGTA (Merck, Darmstadt, Germany). After washing, the samples were labeled with 1 μM AlexaFluor^TM488 Phalloidin (Invitrogen, Waltham, MA, USA) ASB-EGTA without Triton X-100 for 30 min in the dark. Finally, labeled pollen tubes were transferred to microscope slides and covered with ProLong Gold mounting medium (Life Technologies, Carlsbad, CA, USA), and images of microfilaments were acquired using the software package LAS AF connected to a Leica SP8 confocal microscope with a 63× (numerical aperture, 1.4) Plan Apochromat DIC H immersion oil lens. For image processing and analysis, ImageJ (NIH, Bethesda, MD, USA) software was used.

In vitro elongated WT and si1PhCRT pollen tubes (cultivated about 4–5 h) were transferred to liquid culture medium containing 400 μM m-maleimidobenzoyl-N-hydroxysuccinimide ester (Sigma-Aldrich) for 6 min. Next, pollen tubes were permeabilized with an actin-stabilizing buffer (ASB) composed of 100 mM PIPES, 5 mM MgSO₄, 0.5 mM CaCl₂, and 0.05% Triton X-100, pH 9.0, for 5 min, and fixed with freshly prepared 2% formaldehyde in ASB at room temperature for 30 min. Pollen tubes were washed three times with ASB as above except at pH 7.0 and supplemented with 10 mM EGTA (Sigma-Aldrich). After washing, the samples were labeled with 1 μM AlexaFluor^®488 Phalloidin (Molecular Probes) ASB-EGTA without Triton X-100 for 30 min in the dark. Finally, labeled pollen tubes were transferred to microscope slides and covered with MobiGLOW mounting medium and images of microfilaments were acquired using the software package LAS AF connected to a Leica SP8 confocal microscope with a 63× (numerical aperture, 1.4) Plan Apochromat DIC H immersion oil lens.