A home-built setup was used for STED-anisotropy measurements. CW laser at 488 nm, generated by an OBIS LX 120 mW CW laser (Coherent), was passed through Glan–Taylor polarizer (Thorlabs) to obtain highly pure (100,000:1) linearly polarized excitation beam. CW laser at 592 nm (VFL-P-1000 592; MPB Communications Inc.) was passed through a vortex phase plate (VPP-1a; RPC Photonics) to generate a depletion with a doughnut profile. The excitation and depletion beams were focused on the sample using a 100×, 1.49 NA oil immersion objective (Nikon). Excitation modulation and synchronous detection were carried out according to SI Appendix, section 3 .
The emitted fluorescence was collected by the same objective lens. The parallel and orthogonal components of the emitted fluorescence were separated by a polarizing cube (CCM1-PBS251; Thorlabs) and steered to 2 single-photon counters (SPCM-AQRH; EXCELITAS) for anisotropy measurements. The TTL signal from SPC was acquired with a DAQ card (PCIe-6353; National Instruments). Data acquisition and analysis was automated by custom LabVIEW software (National Instruments).
The emitted fluorescence was collected by the same objective lens. The parallel and orthogonal components of the emitted fluorescence were separated by a polarizing cube (CCM1-PBS251; Thorlabs) and steered to 2 single-photon counters (SPCM-AQRH; EXCELITAS) for anisotropy measurements. The TTL signal from SPC was acquired with a DAQ card (PCIe-6353; National Instruments). Data acquisition and analysis was automated by custom LabVIEW software (National Instruments).