Easy3d module

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

We used a custom-built STED/confocal setup^{55 (link)} constructed around an inverted microscope body (DMI 6000 CS, Leica Microsystems) which was equipped with a TIRF oil objective (x100, 1.47 NA, HXC APO, Leica Microsystems) and a heating box (Cube and Box, Life Imaging Services) to maintain a stable temperature of 32 °C. A pulsed-laser (PDL 800-D, PicoQuant) was used to deliver excitation pulses at 488 nm and a de-excitation laser (Onefive Katana 06 HP, NKT Photonics) operating at 594 nm was used to generate the STED light pulses. The STED beam was profiled to a donut shape using a spatial light modulator (Easy3D Module, Abberior Instruments). Image acquisition was controlled by the Inspector software (Abberior Instruments). The spatial resolution of the microscope was 175 nm (x-y) and 450 nm (z) in confocal mode and 60 nm (x-y) and 160 nm (z) in STED mode.

We used a home-built 3D-STED setup (for details, see Inavalli et al., 2019 (link)) constructed around an inverted microscope body (DMI 6000 CS, Leica Microsystems), which was equipped with a TIRF oil objective (100×, 1.47 NA, HXC APO, Leica Microsystems) and contained within a heating box (Cube and Box, Life Imaging Services) to maintain a stable temperature of 32°C. A pulsed-laser (PDL 800-D, PicoQuant) was used to deliver excitation pulses (90 ps at 80 MHz) at 485 nm and a synchronized de-excitation laser (Onefive Katana 06 HP, NKT Photonics) operating at 592 nm was used to generate the STED light pulses (500–700 ps). The STED beam was reflected on a spatial light modulator (SLM, Easy3D Module, Abberior Instruments) to generate a mixture of doughnut-shaped and bottle-shaped beams for 2D and 3D-STED, respectively. Image acquisition was controlled by the Imspector software (Abberior Instruments). The performance and spatial resolution of the microscope was checked and optimized by visualizing and overlapping the PSFs of the laser beams using 150-nm gold nano-spheres and 40-nm fluorescent beads and correcting the main optical aberrations with the SLM. The spatial resolution was 175 nm (lateral) and 450 nm (axial) in confocal mode and 60 nm (lateral) and 160 nm (axial) in STED mode.