Blp01 647r 25

Super-resolution dSTORM measurements were performed on a custom-made inverted microscope based on a Nikon Eclipse Ti-E frame (Nikon Instruments Europe BV, Amsterdam, The Netherlands). After being conditioned (through spatial filtering via fiber coupling and beam expansion), the applied laser beams were focused onto the back focal plane of the microscope objective (Nikon CFI Apo 100×, NA = 1.49), which produced a collimated beam on the sample. All dSTORM images were captured with a linearly polarized beam and EPI illumination at an excitation wavelength of 647 nm (MPB Communications Inc., Pointe-Claire, QC, Canada: 647 nm, P_max = 300 mW). The laser intensity was controlled via an acousto-optic tunable filter (AOTF). Images were captured by an Andor iXon3 897 EMCCD camera (Andor: Belfast, UK; 512 × 512 pixels with 16 μm pixel size). Frame stacks for dSTORM super-resolution imaging were typically captured at a reduced image size (crop mode). Excitation and emission wavelengths were spectrally separated with a fluorescence filter set (Semrock, Rochester, NY, USA; LF405/488/561/635-A-000) and an additional emission filter (Semrock, BLP01-647R-25) in the detector arm. During the measurements, the perfect focus system of the microscope was used to keep the sample in focus with a precision of <30 nm.

Lung tissue slices were imaged on a ctASLMv2^{118 (link)} microscope chamber controlled by navigate¹¹⁹ . Nuclei were imaged using the TO-PRO-3 647 via illumination with a LuxX 642 nm, 140 mW at 100% laser power and a Semrock BLP01–647R-25 filter in the detection path. Cancer cells were imaged via illumination with a LuxX 488–150, 150 mW at 100% laser power and a Semrock FF01–515/30–32 bandpass filter in the detection path. Images were acquired with a Hamamatsu ORCA-Flash 4.0 v3 with 200 ms integration time in lightsheet readout mode.