Celltirf 4line system

All fluorescence microscopy experiments were performed on an Olympus IX 83 motorized inverted fluorescence microscope equipped with the CellTIRF-4Line system, a back-illuminated electron-multiplying charge-coupled device camera (Andor), Sutter excitation and emission filter wheels under the control of the CellSens Dimension software. Wide-field fluorescence microscopy images were acquired using a 100× UPlanSApo 1.4 numerical aperture (NA) objective lens, an EXFO X-Cite Series 120 light source, an Olympus MT20 filter set for 4′,6-diamidino-2-phenylindole, EGFP, and Tetramethylrhodamine (TAMRA), and a Chroma filter set (ET620/60x, ET700/75m, and T660lpxr) for Qusar670. Three-dimensional (3D) image stacks were acquired with 0.25-μm increments in the z direction, and the image stacks were processed using AutoQuant deconvolution software (Media Cybernetics) and then by Fiji to create a maximum intensity projection image. TIRF imaging and PALM imaging were performed using a 100× 1.46 NA total internal reflection objective with 405-nm (100 mW), 488-nm (150 mW), 561-nm (150 mW), and 640-nm (140 mW) excitation lasers.

Coverslips (Warner Instruments, 25 mm, #1.5 thickness) were used. C2C12 cells were cultured on cleaned Coverslips coated with fibronectin (2 μg/mL in PBS, pH 7.4; Sigma). After cells were immunostained for MyoD and CTCF (see Immunofluorescent staining section above), they were blocked twice with 1 × PBS solution containing 50 mM glycine for 10 min, washed once in 1 × PBS, and then subjected to dual-color imaging in dSTORM imaging buffer (1 × PBS, 100 mM β-mercaptoethylamine (Sigma, Cat.N: 30070), pH 7.4) containing TetraSpeck beads (Life Technologies, Cat.N: T7279), which were used as fiducial markers for x−y drift correction and for overlaying the two-color images.
dSTORM images were acquired on an Olympus IX83 motorized inverted fluorescence microscope equipped with the CellTIRF-4Line system, a 150 × 1.45 NA total internal reflection objective, a back-illuminated EMCCD camera (Andor), an IX3-U-m4TIRSbx filter set (Olympus), and 488 nm (150 mW) and 640 nm (140 mW) lasers. dSTORM images of Alexa 647 and Alexa 488 were acquired sequentially in that order. All images were acquired at 10 frames per second. Individual molecules were localized and super-resolution images were reconstructed using the ThunderSTORM analysis module available for ImageJ.

Fluorescence microscopy experiments were performed on an Olympus IX 83 motorized inverted fluorescence microscope equipped with the CellTIRF-4Line system, a 100× UPlanSApo 1.4NA objective lens, a back-illuminated EMCCD camera (Andor), Sutter excitation and emission filter wheels under the control of the CellSens Dimension software. Images of DAPI, EGFP/ATTO488 and TAMRA were acquired using the Olympus MT20 filter set for DAPI, EGFP and TAMRA and images of FRET and ATTO647N were acquired using Chroma filter sets (ET545/25x, ET700/75m, T565lpxr) and (ET620/60x, ET700/75m, T660lpxr), respectively, with the excitation light provided by a X-Cite Series 120 light source housing a Mercury Lamp (EXFO). Simultaneous, dual-color images of EGFP and FRET were acquired using IX3-U-m4TIR-Sbx, and a DV2-cube (ET525/50m, 585dcxr, ET655lp, Photometrics), with the excitation light of EGFP and FRET provided by a 488nm laser line (150 mW) and a 561nm laser line (150 mW), respectively. Three-dimensional (3D) image stacks were acquired with 0.25 μm increments in the z-direction. All images were analyzed using Fiji (21 (link)), the AutoQuant deconvolution software (MediaCybernetics), or custom-written MATLAB (Version R2014b 64-bit, Mathworks) programs.