1c resolft quad scanning microscope

RESOLFT nanoscopy was performed using a modified 1C RESOLFT QUAD Scanning microscope (Abberior Instruments, Goettingen, Germany). For RESOLFT imaging of Drosophila melanogaster, the following switching scheme was applied to the sample at each scanning position: First, rsEGFP2 proteins were switched into the on-state with light of 405 nm. Second, rsEGFP2 proteins in the periphery of the focal spot were switched off using a 488 nm doughnut-shaped beam. The 2D-doughnut was realized by using a phase plate, whereas the 3D-doughnut was generated by using a spatial light modulator. Third, on-state fluorophores at the center of the spot were read out with a Gaussian shaped beam of 488 nm light. We introduced a short 5 µs break between each step. The RESOLFT images were recorded with or without line accumulation depending on the signal intensity. A detailed listing of the laser powers, switching times, line accumulations and scanning step sizes of all RESOLFT images shown are provided in Supplementary file 2. The corresponding confocal images were recorded by applying the same switching scheme as used for the RESOLFT imaging without the illumination step using a doughnut shaped beam (step 2).

All images shown were recorded with transiently
transfected HeLa cells 24 h post-transfection. Cells were mounted
in DMEM without phenol red (Thermo Fisher) and imaged at ambient temperature
with a customized 1C RESOLFT QUAD scanning microscope (Abberior Instruments,
Göttingen, Germany). The microscope was equipped with an UPLSAPO
1.4 NA 100× oil immersion objective (Olympus, Shinjuku, Japan)
as well as 405 and 488 nm continuous-wave lasers (both Cobolt, Solna,
Sweden). The 405 nm doughnut-shaped beam was realized with an easy
3D module (Abberior Instruments). Fluorescence was detected with a
SPCM-AQRH-13 photon counting module (Excelitas Technologies, Waltham,
MA, USA) with a HC 550/88 detection filter. Laser powers were measured
behind the objective with a PM200 power meter with the S170C sensor
(ThorLabs, Newton, NJ, USA). The circular or ring-like area of both
beams at FWHM intensity in the focus were determined and used for
further calculations. Images and filament intensity line profiles
measured with three adjacent lines were analyzed with the Fiji distribution
of ImageJ (v1.52p)^{58 (link),59 (link)} and OriginPro 2018b (OriginLab).
This manuscript has been previously submitted to the preprint server
bioRxiv.⁶⁰