Proyecto conjunto de análisis de imágenes de Yossmayer iniciado 11-2023
Métodos del paper original
Confocal laser scanning microscopy and image analysis Plants and recombinant HyPer7 were imaged in a confocal laser scanning microscope (Zeiss LSM 780, connected to an Axio Observer.Z1; Carl Zeiss Microscopy, Jena, Germany) using a 5x lens (EC Plan-Neofluar 5x/0.16) or a 40x lens (C-Apochromat 40x/1.2 W Korr). Samples were mounted in imaging buffer (10 mM MES, 10 mM MgCl2, 10 mM CaCl2, 5 mM KCl, pH 5.8). All sensors were sequentially excited at 488 nm and 405 nm, and the fluorescence emission was collected at 508–535 nm. The laser power was adjusted according to the respective relative excitation efficiencies on the two excitation peaks of the respective probes. Imaging was performed without averaging with a pixel dwell time of 1.58 µs/pixel. Samples were scanned in areas set to 235.7 µm x 235.7 µm, 124.8 µm x 124.8 µm or 60.7 µm x 60.7 µm. Hydrogen peroxide (H2O2) and dithiothreitol (DTT) perfusion experiments were performed as described in (Ugalde et al., 2020) using a RC-22 perfusion chamber mounted on a P1 platform (Warner Instruments, Hamden, CT). To inhibit photosystem II (PSII), samples were pre-treated for 45 min with 20 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the dark (SigmaAldrich, Steinheim, Germany) dissolved in ethanol. The fluorescence ratio was calculated as 405 nm / 488 nm for roGFP2-Orp1 and 488 nm / 405 nm for HyPer and HyPer7 and normalized to the ratio values at the start of the experiments (t = 0 min). Ratiometric imaging and analysis of single plane images was performed with a custom-written MATLAB script (Fricker, 2016) or FIJI (Schindelin et al., 2012).
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++= 20203-12-14
Armo una imagen de prueba en fiji dividiendo Canal 2 (488)/ Canal 1 (405). Paso todas las im'agenes a 32 bit. Uso LUT Royal para visualizar.