Assembly and characterization of the SMT/mSIM microscope

To acquire SMT movies while collecting reference images of the nuclear architecture, we custom-built a microscope capable of both single-molecule imaging of NFs and super-resolved mSIM of a reference channel. The scheme of our SMT/mSIM microscope is depicted in Supplementary Fig. ^1a. Briefly, the microscope is composed by two illumination arms, connected to a commercial microscope Frame (Olympus IX-71, Olympus Life Sciences). The first illumination arm, directs the laser light from both a 200 mW 561 nm laser (Cobolt 06-DPL, Hubner Photonics) and a 200 mW 647 nm laser (Coherent Obis, Coherent Inc) to perform SMT using HILO illumination. Here, a movable mirror (MM) in a conjugated plane of the back focal aperture of the objective allows for achieving the desired light beam inclination in the object plane (roughly 67°). The second line expands the collimated light from a 405 nm and a 488 nm lasers (Coherent Obis, Coherent Inc.) to 0.5 cm in size and directs it onto a DMD (Vialux v7000) which creates a pattern of diffraction-limited spots on the sample. The physical size of each DMD micromirror is 13.67 μm and, with the lenses used in our microscope (see scheme in Supplementary Fig. ^1a), this corresponds to a projected image of each pixel on the sample plane of ~117 nm. The chosen illumination pattern (an equilateral triangular lattice with side equal to 16 DMD pixels) is scanned over the mSIM field of view. 224 different images are necessary to completely scan the entire field of view. The two illumination arms are next combined through a dichroic mirror (DM)(Di03-R488-t3-25 × 36, Semrock Inc.) that directs the excitation light to the sample through a quad-band dichroic (Di03-R405/488/561/635-t3-25 × 36, Semrock inc.) and a 60 × 1.49NA oil-immersion objective (Olympus ApoN 60 × 1.49 Oil, Olympus Inc.). The fluorescent light originating from the sample is collected by the same objective, filtered by the quad-band dichroic and a quad-band emission filter (FF01-466/523/600/677-25, Semrock Inc.) and directed to a sCMOS camera (Orca Fusion C14440-20UP, Hamamatsu Photonics) resulting in an image pixel size equal to 108.3 nm. The microscope is equipped with control systems for temperature (37 °C), CO₂ (5%), and humidity, to maintain cells under physiological conditions during live-cell experiments.

We verified that upon reconstruction of the mSIM super-resolved image (see below), the lateral resolution of the microscope increased by a factor higher than 1.4× (Supplementary Fig. ^1b), and that minimal chromatic aberration (below the microscope resolution limit, Supplementary Fig. ^1c) was present when imaging subdiffraction fluorescent beads with the two different illumination arms of the microscope. Reconstruction of the mSIM image was performed by custom-written routines in Matlab (available at https://github.com/shiner80/Recon_mSIM), as described in Supplementary Note ¹.