A1r confocal scanhead

Correlative data were acquired with a commercial inverted two-layer Nikon Eclipse Ti2 microscope, equipped with an A1R confocal scanhead and N-STORM imaging module (Nikon instruments, Tokyo, Japan) and controlled by the NIS Elements software (version 5.30.02). A Nikon CFI SR Apochromat TIRF 100× oil, 1.49 NA objective was used for all the measurements. Confocal images were scanned in galvanometric mode and the ROI size was set to 1024 × 1024 pixels, with a pixel size of ∼0.07 μm. The scanning pixel dwell-time and laser power were set to avoid the photobleaching effect. Confocal and widefield (dSTORM) images were aligned targeting the position of FNDs using the alignment routine of NIS Elements software (version 5.30.03, Nikon instruments, Tokyo, Japan) (Pelicci et al., in preparation).

All data were acquired with a commercial Nikon Eclipse Ti2 inverted microscope (Nikon instruments, Tokyo, Japan), equipped with A1R confocal scanhead, N-SIM and N-STORM modules (Nikon instruments, Tokyo, Japan). The fully motorized automated microscope was controlled by the NIS Elements software (version 5.42.01, Laboratory Imaging s.r.o, Praha, Czech Republic). The system performs multicolor widefield, confocal, and single-molecule localization imaging thanks to a pE-4000 (CoolLED, Andover, UK) light source with 16 selectable LED wavelengths (Widefield microscopy) and a LU-NV laser unit (Nikon instruments, Tokyo, Japan) equipped with 5 laser lines (405 nm (23.1 mW), 440 nm (25.5 mW) 488 nm (79.1 mW), 561 nm (79 mW), and 647 nm (137 mW)) (Confocal, SIM, TIRF and dSTORM microscopy). The microscope is equipped with fluorescence filter sets for the optimal detection of the employed fluorochromes thanks to a double-layer turret allowing the combination of 5 cubes per layer. The upper turret is devoted to widefield and STORM imaging, with the lower one acting as an additional filter wheel. SIM images were instead collected through the lower layer by spatially calibrated filter cubes. Emitted light was collected by a CMOS camera (Dual ORCA Flash 4.0 Digital CMOS camera C13440, Hamamatsu, Tokio, Japan) set on a 16-bit scale detection modality (Widefield/SIM/TIRF/dSTORM).

All data were acquired with a commercial inverted Nikon Eclipse Ti2 microscope (Nikon instruments, Tokyo, Japan), equipped with an A1R confocal scanhead and N-SIM and N-STORM modules (Nikon instruments, Tokyo, Japan). The fully motorized automated microscope was controlled by the NIS Elements software (version 5.42.01). The system performed multicolor widefield, confocal, and single-molecule localization imaging thanks to (i) an LED light source (pE-4000, CoolLED, Andover, UK) with 16 selectable wavelengths for widefield microscopy; (ii) a laser unit (LU-NV, Nikon instruments, Tokyo, Japan) equipped with 5 laser lines (405 nm (23.1 mW), 440 nm (25.5 mW) 488 nm (79.1 mW), 561 nm (79 mW), 647 nm (137 mW)) for confocal microscopy, and (iii) a laser bench (L4Cc combiner, Oxxius S.A., Lannion, France) equipped with four high-power sources (405 nm (216 mW), 488 nm (240 mW), 561 nm (240 mW), 640 nm (360 mW)) and two acousto-optic modulators for single-molecule microscopy). Emitted light was filtered by a filter wheel (Optospin, Cairn Research Ltd., Faversham, Kent, UK) and then was collected by a CMOS camera (Dual ORCA Flash 4.0 Digital CMOS camera C13440, Hamamatsu, Tokyo, Japan) set on a 16-bit scale detection modality (Widefield/DNA PAINT).