Tcs sp8 aobs confocal laser scanning microscope

FRAP experiments were carried out with a Leica TCS SP8 AOBS confocal laser scanning microscope (HCX PL APO 63×/1.2 water objective, PMT detector). Rubisco holoenzyme was labeled with Alexa Fluor 532 NHS ester (Rubisco_AF5, ThermoFisher) (~3.6 dye molecules bound per Rubisco holoenzyme), and other proteins were labeled as described above. Reactions (20 μl) in buffer D (50 mM Tris pH 8.0/100 mM KCl/10 mM Mg(OAc)₂) in the presence or absence of 5 mM DTT, and proteins at the concentrations stated in figure legends, were combined. After incubation for 5 min at 25 °C, reactions were transferred to an uncoated chambered coverslip (μ-Slide angiogenesis, Ibidi) followed by incubation for a further 15 min before analysis. Images before and 10 min after photobleaching were recorded in a single focal plane at 5-s time intervals. Bleaching was performed with a bleach point model using either a 405-nm diode laser at 2% intensity or a 532-nm argon laser at 100% intensity in one repeat, with a dwell time of 100 ms. The software Fiji was used for image analysis^{62 (link)}. Proteins from the same purification batches were analyzed repeatedly.

The confocal imaging was performed at the Imaging Facility of Max Planck Institute of Biochemistry, Martinsried, on a LEICA (Wetzlar, Germany) TCS SP8 AOBS confocal laser scanning microscope equipped with a LEICA HC PL APO 63x/NA1.4 oil immersion objective or on a ZEISS (Jena, Germany) LSM780 confocal laser scanning microscope equipped with a ZEISS Plan APO 63x/NA1.46 oil immersion objective. Leica LAS AF and Zeiss ZEN 2011 software packages were used, respectively. Image analysis was performed manually by using ImageJ or automated by employing a custom-made Fiji script.

Worms were placed in a 5 μl drop of 1 mM levamisole on a 4% agarose pad. After 30 min, a coverslip was placed over the worms. Images were collected using a Leica TCS SP8 AOBS confocal laser scanning microscope. Images were analysed using ImageJ software (Bethesda, MD, USA).