N sim system

For the super-resolution imaging of mitochondria, BMDMs were incubated in the completed medium containing 100 nM MitoTracker Red CMXROS for 30 min. After incubation, cells were washed twice and replaced with the completed medium for live-cell imaging. Super-resolution images were acquired using a Nikon N-SIM system (Nikon, Japan) with an oil-immersed 100× and 1.49 NA objective. Images for live-cell imaging (live N-SIM) were taken at a single Z-plane. Images were captured using Nikon NIS-Elements and reconstructed using slice reconstruction in NIS-Elements. Cells used for live-cell imaging were maintained in a 37 °C, 5% CO_2, and humidified environment.
For super-resolution imaging of single liposome, MitoTracker-loaded liposome and Matrigel were mixed and smeared on the confocal dish to immobilize the liposome. Liposomes were imaged with the same imaging conditions used in live-cell experiments.

M–L contact sites in both fixed and live neurons were imaged using super-resolution SIM. For SIM analysis, neurons were cultured on nitric-acid-treated, PDL/laminin-coated High Precision Glass Cover Slip (Bioscience Tools, #CSHP-No1.5-12). Samples were prepared using the same protocol for regular ICC. Super-resolution images were taken on a Nikon N-SIM system with a 100× oil immersion objective lens, 1.49 NA (Nikon). Images were captured and reconstructed using Nikon NIS-Elements.

3D-SIM images were acquired by N-SIM system (Nikon). We performed 3D nucleus segmentation on the reconstructed 3D-SIM image via the following four steps: (1) Background was removed via a 3D median filter with a radius of 5 µm to estimate the local background caused by autofluorescence, which was subtracted from the SIM image. (2) To identify each 3D nucleus along the axial direction, we track the peak intensity distribution along the axial center position of the nucleus. (3) In the initial nucleus segmentation, at the axial position with the maximum intensity, the region of the nucleus was first manually selected by tracking the border of the nucleus, in which the central position was calculated. The precise border of each nucleus was then adaptively defined by finding the fastest changing gradient along each radial direction. This process was repeated for all axial positions above and below the central plane, for 3D nuclei segmentation. Finally, to calculate DNA occupancy, the voxels with an intensity larger than three times of square root of the background were recognized as the regions with valid signals. DNA occupancy was defined as the percentage of voxels with valid signals over the total number of voxels for each 3D nucleus.

The agarose pads with induced MG1665 cells were prepared similar to those in the “Imaging the IbpA protein with wide-field fluorescence microscopy” section. SIM images were acquired on a Nikon N-SIM system equipped with a Nikon SR HP Apo TIRF 100X 1.49NA objective, a Hamamatsu ORCA-Flash4.0 camera (65 nm per pixel), and 488 nm and 561 nm lasers from a Nikon LU-NV laser launch. Cells were identified using DIC to avoid photobleaching. For each 2D-SIM image, nine images were acquired in different phases via the built-in 2D SIM modes. Super-resolution image reconstruction was performed using the Nikon Elements SIM module.

SIM images were taken with an N-SIM system (Nikon) attached to a Ti2-E inverted microscope (Nikon) with a CMOS camera (ORCA-Flash 4.0 V3; Hamamatsu Photonics) using a Plan Apochromat 100×/1.35 NA silicon-immersion objective lens at a step size of 0.12 μm. The chromatic aberration of the system was calibrated and corrected by 0.1 μm TetraSpeck beads (T7279; Thermo Fisher Scientific) in the mounting medium. Images were reconstructed and analyzed using NIS elements AR (Nikon) according to the manufacturer's protocol.

SIM super-resolution images were taken on a Nikon N-SIM system with a 100× 1.49-NA oil-immersion objective lens (Nikon). Images were captured using NIS-Elements (Nikon) at 1 frame every 7 s and reconstructed using slice reconstruction in NIS-Elements (Nikon). Images for live-cell imaging (live N-SIM) were taken at a single z-plane. Cells used for live-cell imaging were maintained in a temperature-controlled chamber (37°C) at 5% CO₂ in a TokaiHit stagetop incubator.