Tcs sp2 aobs laser scanning microscope

Chlorophyllian cells were observed with a Leica TCS SP2-AOBS laser scanning microscope (Leica Microsystems). Fluorescence excitations were obtained with the 488nm line of an argon laser, and fluorescence emissions were filtered between 545 nn and 565nm and between 635nm and 655nm to record green and red fluorescence intensities, respectively. Images were acquired with a HCPL Apochromat CS ×63 (N.A. 1.40) oil immersion objective to allow for ratiometric processing.

Labelled cells were deposited between the slide and the cover slip and observed with a Leica TCS SP2-AOBS laser scanning microscope (Leica Microsystems) coupled to a HCPL Apochromat CS 63× (N.A. 1.40) oil immersion objective. Fluorescence excitations were obtained using either the 543-nm line of a helium-neon laser (rhodamine-phalloidin), the 488-nm line of an argon laser (tubulin tracker), or a 405-nm diode (calcofluor). Fluorescence emissions were recorded between 555–700 nm (rhodamine-phalloidin), 500–600 nm (tubulin tracker), and 410–480 nm (calcofluor). For di-4-ANEPPDHQ observation, after excitation at 488 nm, emission intensities were acquired between 540–560 nm (green image) and between 650–670 nm (red image). Ratiometric imaging was performed using the ImageJ software (http://imagej.nih.gov/ij/).

di-4-ANEPPDHQ-labelled leaves were observed as described in (Gerbeau-Pissot et al., 2014 (link)) with a Leica TCS SP2-AOBS laser scanning microscope (Leica Microsystems, Germany) and a HCPL Apochromat CS 63x (N.A. 1.40) oil immersion objective. Images were excited with the 458 nm line and the 488 nm line of an argon laser for CFP and di-4-ANEPPDHQ respectively as described in (Gerbeau-Pissot et al., 2014 (link)). Fluorescence emissions were filtered between 465 and 500 nm for CFP. For di-4-ANEPPDHQ, to obtain ratiometric images, we recorded green and red fluorescence between 540 to 560 nm and 650 to 670 nm, respectively. The mean red/green ratio of pixels (RGM) corresponding to either the global membrane, the 10%, the 5% or the 2% of the most intense CFP pixels were compared on each image.