Las af software

STED-FCS measurements were carried out on primary HDLECs labeled with LYVE-1 Fab Abberior STAR Red as above on the Leica SP8 TCP inverted microscope. The master power of the white-light laser was set to 30%. Sample excitation at 633 nm was used at 1–25% (1–25 μW) output power. STED depletion was carried with a pulsed 80-MHz IR laser (755 nm) at laser powers (0–180 milliwatts). STED-FCS measurements were collected onto a single-photon-counting avalanche photodiode (APD; Micro Photon Devices, PicoQuant, Berlin, Germany) in the external port of the microscope. The APD signal was recorded with a time-correlated single-photon-counting (TCSPC) detection unit (Picoharp 300, PicoQuant), which saves the raw photon stream. The TCSPC control software (SymPhoTime, PicoQuant) allows reconstruction of fluorescence lifetime decays as well as fast calculation of FCS data. The recordings were directly controlled by the Leica LAS AF software, which communicates with the PicoQuant SymPhoTime software as an already integrated FCS package in LAS AF.

Confocal FCS measurements were acquired on a Leica SP8 confocal microscope equipped with a HC PL APO 100 × /1.40 NA oil objective. Prior to the FCS measurement, 1G4 TCR Jurkat T cells were loaded with either CellMask Deep Red, or Cholestorol-PEG-KK114 (Honigmann et al., 2014 (link)) at a concentration of 1 μg mL^-1. FCS recordings were directly controlled by the Leica LAS AF software, which communicates with the PicoQuant SymPhoTime (PicoQuant, Berlin, Germany) software and hardware as a pre-integrated FCS package in LAS AF. Fluorescent light was collected onto a single-photon-counting avalanche photo-diode (APD; Micro Photon Devices, PicoQuant) in the external port of the microscope. The APD signal was recorded with a time-correlated single-photon-counting (TCSPC) detection unit (Picoharp 300, PicoQuant). FCS data were fit with a two-dimensional (2D) diffusion model using the FoCuS-point fitting software (Waithe et al., 2016 (link)).