Lu nv

SIM imaging was performed with a N-SIM based on an inverted microscope (ECLIPSE Ti-E, NIKON), equipped with an oil immersion TIRF objective lens (SR Apo TIRF 100 × , N.A. 1.49, NIKON), a laser system consisting of 405, 488, 561, and 640 -nm diode lasers (LU-NV, NIKON), and an EMCCD camera (iXon3 DU-897E, Andor Technology). SIM imaging with this system is based on a previous report^{17 (link)}. Briefly, excitation lasers were coupled to a multimode optical fiber, collimated, and directed to a fused silica linear transmission-phase grating. A shutter in an intermediate pupil plane discarded all diffraction orders except for 0 and ± 1. The three beams were refocused in the back focal plane of the objective lens. The beams produced as diffraction orders + 1 and −1 were focused near the opposing edges of the back focal plane aperture, and the beam produced as order 0 was focused at its center. Three-dimensional data were acquired with five-pattern phases spaced by 2π/5 and three-pattern orientations spaced 60° apart. The acquired images were computationally reconstructed to obtain a high-resolution image with resolutions of ~115 nm in the x- and y-dimensions and ~270 nm in the z-dimension.

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).

Sample were imaged by a commercial inverted Nikon Eclipse Ti2 microscope (Nikon instruments, Tokyo, Japan) equipped with an A1R confocal scan-head and N-SIM and N-STORM modules (Nikon instruments, Tokyo, Japan) and controlled by NIS Elements software (version 5.42.01). For widefield microscopy, an LED light source (pE-4000, CoolLED, Andover, United Kingdom) with 16 selectable wavelengths for widefield microscopy was employed. The light source for confocal imaging was 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)), while a laser bench (L4Cc combiner, Oxxius S.A., Lannion, France) equipped with four high-power sources (405nm (216 mW), 488 nm (240 mW), 561 nm (240 mW), 640 nm (360 mW)) was employed for single-molecule localization experiments. A filter wheel (Optospin, Cairn Research Ltd, Faversham, Kent, UK) was placed in front of a CMOS camera (Dual ORCA Flash 4.0 Digital CMOS camera C13440, Hamamatsu, Tokyo, Japan) to acquire 16-bit scaled images (Widefield/DNA PAINT).