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

In the case of the one-photon system^{23 (link)}, the excitation light was provided by a pulsed diode laser (λ = 485 nm) and the STED light came from a Ti:Sa femtosecond laser emitting at 838 nm, which was converted to a wavelength of 597 nm by an optical parametric oscillator (OPO). A signal from the OPO was used to trigger pulsed diode laser. We used a glycerol objective with a correction collar to reduce spherical aberrations. In the case of the 2P-STED setup, the excitation light was provided by a second Ti:Sapphire laser tuned to 910 nm running at 80 MHz that was synchronized to the OPO pulses. We used water-immersion objectives with long working distances, either from Nikon (CFI Apo 60X W NIR, 1.0 NA, 2.8 mm WD)^{46 (link)} or Olympus (LUMFI 60×, 1.1 NA, 1.5 mm WD)^{45 (link)}. The STED pulses were broadened in time by passing them through glass rods and a long optical fiber (20 m) to ensure effective de-excitation and maximal resolution enhancement. Synchronization and optimal pulse delay were achieved with phase-locked loop electronics.
The STED beam reflected twice on two different surfaces of a single spatial light modulator (SLM) (Easy3D Module, Abberior Instruments, Göttingen, Germany) to generate doughnut and bottle beams for 2D and 3D-STED microscopy. The SLM was conjugated to the beam scanner (Yanus IV, TILL Photonics, Gräfelfing, Germany) via appropriate relay lenses and the scanner was conjugated to the back focal plane of the objective via the scan and tube lens combination (F_scan = 5 cm and F_tube = 25 cm, Leica Microsystems). In the case of 2P-STED microscopy^{47 (link)}, the STED doughnut was created by passing the STED beam through a vortex phase mask, which imposed a helical 2π phase delay on the wave front. Wave plates (λ/2 and λ/4) were used to make the STED light circularly polarized before it entered into the objective.
The excitation and STED beams were precisely co-aligned using a dichroic mirror and a piezo-positioner. Both beams passed through an x–y beam scanner. The fluorescence was de-scanned and focused into a multimode optical fiber connected to an avalanche photodiode operated in photon-counting mode.
For the FRAP experiments, a second Ti:Sa femtosecond laser beam (at λ = 900 nm) was injected into the optical path of the microscope using a 680 nm short-pass dichroic mirror. For two-color imaging, two fluorophores with partially overlapping excitation and emission spectra (e.g., YFP and GCaMP6s) were imaged simultaneously using a single excitation/STED laser beam pair. The emission signal was split by a 514 nm long-pass dichroic mirror, and was sent into separate photodetectors^{48 (link),49 (link)}.