Elyra ps 1 microscope

TIRF images of the substrate-attached membrane region were acquired at a GE DeltaVision Elite system (GE Healthcare Bio-Sciences, Marlborough, MA) based on an OLYMPUS IX-71 inverted microscope, with an Olympus TIRF 100×/1.49 UAPON objective and a PCO sCMOS 5.5 camera. For imaging the cells at high frequency in TIRF mode (Figure 7), a Zeiss Elyra PS.1 microscope (Carl Zeiss Microscopy, Jena, Germany) with an alpha Plan-­Apochromat 63×/1,46 Oil Corr TIRF objective and two PCO pco.edge 4.2 m sCMOS cameras (PCO-Tech, Romulus, MI) was used. For confocal recordings, a Zeiss LSM 780 microscope equipped with a Plan-Apo 63×/NA 1.46 oil immersion objective was used.
Images were analyzed using the image-processing package Fiji (http://Fiji.sc/Fiji) developed by Schindelin et al. (2012) (link) on the basis of ImageJ (http://imagej.nih.gov/ij). Line-scans and point-scans were performed using Fiji. To plot the charts, data were imported in Microsoft Excel sheets.
Images in Figure 1C and Supplemental Video S1 were processed through SRRFs with the plug-in NanoJ-SRRF, a part of the open-source superresolution microscopy image analysis toolbox NanoJ designed by Laine et al. (2019) . The images were acquired at a frame rate of 1/s with an exposure time of 100 ms. Each single image was processed using the following settings for the SRRF algorithm: radiality magnification 5, ring radius 3, axes 6.

For immunofluorescence labeling, chloroplast suspensions were fixed in 4% paraformaldehyde (PFA) in PBS containing 1% dimethyl sulfoxide for 30 min at RT and subsequently rinsed three times for 10 min in 1× PBS. Unspecific binding sites were blocked by incubation of samples in 1× PBS with Tween 20 (PBST) with 2% dry milk for 1 h at RT. Subsequently, primary antibodies against CPSFL1-FLAG (anti-FLAG, Rabbit) and recombinant biotin-labeled TGD2 were added in a dilution of 1:100 to the samples and incubated for 1 h at RT. For PI4P detection, PI4P-specific antibody (from Mouse) were added in a 1:100 dilution. After three further rinses in PBST for 10 min each, AlexaFluor488 fluorescent anti-rabbit secondary antibodies and AlexaFluor405-labeled Avidin conjugates or Alexa Fluor405-labeled anti-mouse secondary antibodies were added in a 1:200 dilution, and samples were incubated for another hour at RT. Following three rinses with PBST and three rinses with PBS for 10 min each, chloroplasts were mounted on Poly-Lysine−coated coverslips using VectaShield mounting medium and observed by superresolution microscopy on a Zeiss Elyra PS.1 microscope (Zeiss).
For visualization of CPSFL1-YFP in protoplasts, protoplasts were mounted on coverslips in 400 mM mannitol, 10 mM CaCl₂, 20 mM KCl, 5 mM EGTA, and 20 mM MES, pH 5.7, and imaged using an inverted Zeiss LSM710.