Imaging

A few microliters of fixed chloroplasts were placed on a microscope slide and covered with a coverslip. The chloroplasts were imaged with a confocal TCS SP8 system from Leica Microsystems equipped with an HC PL APO CS2 63×/1.20 NA water immersion objective and a tunable white light laser (Leica Microsystems). The excitation wavelength was set to 620 nm and detection wavelength was set to 670 to 730 nm to capture chlorophyll fluorescence. Laser intensity was set to 0.1% and the gain adjusted to prevent over exposure. Z-stacks were recorded to image the complete chloroplasts.

The gels were imaged with one of several microscopes. We used a confocal TCS SP8 system from Leica Microsystems equipped with a HC PL APO CS2 63×/1.20 NA water immersion objective and an argon laser. We also used a confocal TCS SP8 system from Leica Microsystems equipped with a HC PL IRAPO 40×/1.10 NA water immersion objective and 2-photon excitation. Last, we used the ZEISS Elyra 7 with Lattice SIM² with a C-Apochromat 63×/1.2 NA water immersion objective at the ZEISS demo-center in Oberkochen. The excitation was set to 488 nm (single photon excitation) or 750 nm (2-photon excitation) and emission to only record ATTO488 signal (500 to 540 nm). The intensity of the staining was variable between samples, and therefore the gain was kept at 100% and the laser intensity (2.0% to 5.0%) and line accumulation (2 to 8) varied. De-enveloped chloroplasts were selected for imaging. De-enveloped chloroplasts were distinguished from enveloped chloroplasts by the staining intensity outside the grana. Intact chloroplasts had a higher intensity outside the grana, probably due to the presence of Rubisco, while de-enveloped chloroplasts had a higher intensity staining of the grana than their surroundings. We selected de-enveloped chloroplasts for imaging because it enabled the use of an automated detection software for the grana. Z-stacks were recorded to image complete thylakoid network. A SIM² image reconstruction algorithm from ZEISS was applied on the images from the Elyra 7 with Lattice SIM². All 3 microscopes recorded mirror images, so the recorded images were mirrored back before image analysis.