Tetraspeck fluorescent microsphere

Slide-mounted TetraSpeck fluorescent microspheres (T14792, Invitrogen) were imaged before and after data acquisition. Short movies (100 frames) of the fluorescent broadband beads in all three channels were registered in Fiji/ImageJ2 (v.2.3.0/153q) using the descriptor-based series registration (2d/3d + t) Fiji plugin^{61 (link)}. For more than two-channel bead registration, the frames have to be set as channels (in Fiji: Image > Hyperstacks > Re-order Hyperstack). Sub-pixel accurate registration was achieved via Gaussian mask localization fitting of spots, which were used to compute affine (2D) transformation models^{62 (link)}. Since unbiased noise distribution is essential for accurate single particle identification and tracking, the Fiji plugin now allows for nearest-neighbor interpolation that does not change actual pixel intensities and thereby preserves the original noise distribution (as compared to linear or other higher-order interpolation schemes). This update is available via the Fiji Updater (http://fiji.sc/Downloads). It can be found under Plugins > Registration > Descriptor based Registration (2d/3d) and Plugins > Registration > Descriptor based Series Registration (2d/3d + t).

SIM was performed on a commercially developed Zeiss Elyra PS.1 inverted microscope using a Zeiss 63x oil objective lens (NA: 1.4) and pco.edge CMOS camera and ZEN Black software (Zeiss) as described previously [78,79]. Images were captured using SIM paradigms (34-μm grating, 3 rotations and 5 lateral shifts) and processed using the SIM reconstruction module within ZEN Black with default theoretical PSF and other settings. Shifts between acquired channels were corrected for using 100-nm Tetraspeck fluorescent microspheres (Invitrogen, T7279).

The images were registered and analyzed by Fiji^{83 (link)} and Mathematica (Wolfram) software. To achieve subpixel registration accuracy, parameters for shifting, scaling, and rotating camera images were determined by the least-squares fitting of fluorescent bead images (100 nm TetraSpeck fluorescent microspheres, Invitrogen). The experimental data from each channel were processed through an affine transformation and overlapped in false-color channels for visualization. The locus trajectory was obtained by tracking the locus position over time, and graphs were generated by OriginPro (OriginLab version 2019b).

Images were registered and analyzed using Fiji (Schindelin et al., 2012 (link)) and Mathematica (Wolfram) software. Images obtained by using green and red channels were registered by 0.1 µm coverglass-absorbed TetraSpeck fluorescent microspheres (Invitrogen) as a standard sample. To eliminate movement from live cells, the localization of individual genomic loci was calibrated by the motion relative to the nuclear centroid.

To analyse the substructural organization of RNAPII molecules beyond the classical Abbe-Rayleigh limit of ~250nm, SIM was applied that yields a 2-fold improvement in all spatial directions. Coverslips bearing the labelled nuclei were placed into Chamlide™ magnetic chambers (Live Cell Instrument, South Korea) and submerged in phosphate-buffered saline (PBS; pH 7.5) supplemented with 1% β-mercaptoethanol prior to SIM imaging on a Zeiss ELYRA PS.1 microscope (Carl Zeiss Microscopy, Germany) equipped with a Plan-Apochromat 63×1.4 oil objective. Optimal grid sizes for each wavelength were chosen according to the recommendations of the manufacturer. For 3D-SIM, stacks with a step size of 110nm were acquired sequentially for each fluorophore starting with the highest wavelength dye. The centre of the stack was chosen to coincide with the main plain along the axis of the ellipsoidal nuclei to allow the alignment of SIM and PALM images. The correction of chromatic aberrations was performed with the ZEN Channel alignment tool using a template obtained from imaging TetraSpeck fluorescent microspheres (200nm in diameter; InVitroGen) and affine correction. Thus, the corrections achieved a precision of <100nm.

Daudi B cells were washed in PBS once then stained in 2% FBS/PBS at a concentration of 1 × 10⁶ cells/ml with 10 μg/ml of Alexa Fluor® 647 labeled pinatuzumab IgG and 2 μg/ml Alexa Fluor® 488 labeled anti-human IgM Fab fragment for 20 min at 4°C. Cells were washed 2 times with PBS and resuspended in PBS. Cells were incubated at 37°C for 5 min before injecting in FSC2 chambers preheated to 37°C and allowed to spread for 10 min. Chambers were gently flushed with PBS to wash unbound cells and cells were fixed with 4% PFA and 0.2% glutaraldehyde in PBS for 40 min at room temperature. Chambers were washed 3 times with PBS. Prior to image acquisition, chambers were incubated with dSTORM imaging buffer containing 0.1 M β-mercaptoethylamine (MEA, Sigma-Aldrich), 0.5 mg/ml glucose oxidase, 40 μg/ml catalase, and 10% glucose in PBS. For dual-color dSTORM, samples were incubated in PBS containing 0.1 M MEA, 3% (v/v) OxyFlourTM (Oxyrase Inc.), 20% (v/v) of sodium DL-lactase solution (L1375, Sigma-Aldrich) adjusted to pH ~8.3. Fiducial markers (100 nm Tetraspeck Fluorescent Microspheres, Invitrogen) were added to buffer and allowed to settle for 5 min prior to imaging.