DOI: 10.5061/dryad.tx95x6b6w
title: Decoding the molecular interplay of endogenous CD20 and therapeutic antibodies with fast volumetric nanoscopy

description: Elucidating the interaction between membrane proteins and antibodies
 requires fast whole-cell imaging at high spatiotemporal resolution.
 Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but
 suffers from limited spatial resolution. DNA-PAINT achieves molecular
 resolution but is practically restricted to two-dimensional imaging due to
 long acquisition times. Here, we introduce two-dye imager (TDI) probes,
 manifesting negligible background and amplified fluorescence signal upon
 transient binding, enabling ~15-fold faster imaging. Using a combination
 of TDI-DNA-PAINT and LLS microscopy on B cells, we reveal the oligomeric
 states and interaction of endogenous CD20 with type I and II therapeutic
 monoclonal antibodies (mAbs) rituximab (RTX), ofatumumab (OFA), and
 obinutuzumab (OBZ), respectively, unperturbed by surface effects. Our
 results demonstrate that CD20 is abundantly expressed on microvilli that
 are concatenated by mAb binding accompanied by a concentration-dependent B
 cell polarization and stabilization of microvilli protrusions. These
 findings, we believe, will aid rational design of improved immunotherapies
 targeting tumor-associated antigens.
description: <strong>Ensemble absorbance and fluorescence spectroscopy.
 </strong>UV/visible absorbance spectra and steady-state fluorescence
 emission spectra of fluorescently labeled imager probes were recorded
 using a Jasco V650 absorbance spectrometer and Jasco FP-6500
 spectrofluorometer respectively. For absorbance spectra, 500 nM Ox2 and
 Ox1 imagers were used (fig. S1). In case of fluorescence spectra, 1 nM
 imager strands and respective docking strands were used in
 10<strong><sup>4</sup></strong> molar excess (10
 µM). Fluorescence emission spectra of P3 labeled with 2×ATTO655, 2×Cy5,
 and 2×TMR, 2×ATTO520 R4 (fig. S2), and 2×Ox2-R4 imager strands (fig. S3A).
 Samples were measured in a 3 mm path-length cuvette (Hellma, 105.251-QS)
 in PBS (Sigma-Aldrich, #D8537-500ML) containing 500 mM NaCl adjusted at pH
 7.4. <strong>Fluorescence correlation
 spectroscopy (FCS). </strong>FCS measurements on Ox2-P3 probes (Fig.
 1C and fig. S3B-C) were carried out using a commercial time-resolved
 confocal fluorescence microscope setup (MicroTime200, PicoQuant) as
 described previously<strong>
 </strong><em>(42)</em>. Imager strands were used at a
 concentration of 1 nM dissolved in PBS (Sigma-Aldrich, #D8537-500ML)
 containing 500 mM NaCl adjusted at pH 7.4, while the complementary docking
 strand was 10<sup>4 </sup>times higher i.e., 10 µM.
 <strong>Single color TDI-DNA-PAINT imaging.
 </strong>TDI-DNA-PAINT imaging of microtubules in COS7 cells (Fig.
 1D), DNA origami structures (fig. S4D-G), and CD20 in Raji cells (Fig. 3B,
 fig. S8-S11) were performed utilizing an inverted wide-field fluorescence
 microscope (IX-71; Olympus). For excitation of Ox2 probes, a 640 nm diode
 laser (Cube 640-100C, Coherent) in combination with a clean-up filter
 (laser clean-up filter 640/10, Chroma) was used. The laser beam was
 focused onto the back focal plane of the oil-immersion objective (100×, NA
 1.51; Olympus). Emission light was separated from the illumination light
 using a dichroic mirror (HC 560/659; Semrock) and spectrally filtered by a
 band-pass filter (FF01-679/41-25, Semrock). Images were recorded with an
 electron-multiplying CCD camera chip (iXon DU-897; Andor). Pixel size for
 data analysis was measured to be 104 nm. For classical DNA-PAINT and
 TDI-DNA-PAINT experiments on cellular microtubule networks, 5 nM Ox2-P3 /
 Ox2-R4 probe and 100 pM Ox1-P3 probe was added to the respective
 glass-bottomed chamber wells containing COS7 cells fixed and immunolabeled
 with corresponding MetTet-P3’ docking strand (Fig. 1D). For measurements
 on Raji cells on CD20 labeled via RTX or 2H7, 5 nM Ox2-R4 probe was used.
 Blinking movies (Supplementary movies S1-S12, S18-S21) were recorded for
 representative 128 × 128-pixel areas (for microtubule in COS7 cells) and
 256 × 256-pixel areas or similar (for CD20 with RTX or 2H7 in Raji cells)
 with TIRF excitation with an exposure time of 100 ms (frame rate 9.96 Hz)
 and irradiation intensity of 0.8 kW/cm<sup>2</sup>. Images
 were recorded for 18k frames (30 min) for each dataset (Table S2). We
 recorded images of DNA-origami samples using a different setup but with
 same optical parameters and components as for the previous one. We used
 this second setup as it is a more robust “drift-free” system in our
 laboratory and can be beneficial for origami samples. We added 25 nM of
 Ox2-R4 probes to the well containing DNA-origami nanostructures labeled
 with MetTet -7×R4’ docking strand incubated in the imaging buffer for
 origami. Images were recorded for 128 × 128-pixel areas (Movie S3, Table
 S2) with exposure times of 30 ms for 90k frames at 1.1
 kW/cm<sup>2</sup>. Pixel size for data analysis was measured
 to be 128 nm. <strong>Dual color TDI-DNA PAINT
 imaging. </strong>All two-color TDI-DNA-PAINT measurements were
 performed on an inverted wide-field fluorescence microscope (IX-71;
 Olympus) equipped with an oil-immersion objective (60×, NA 1.49; Olympus),
 a nose-piece stage (IX2-NPS, Olympus), a 514 nm and 639 nm lasers (Genesis
 MX STM-Series, Coherent) and a suitable dichroic mirror (R442/514/635,
 Chroma). The fluorescence emission of ATTO Oxa14 and ATTO520 coupled
 imager strands were acquired simultaneously and projected on two
 electron-multiplying charge-coupled device (EMCCD) cameras (iXon Ultra
 897, Andor) by means of a dichroic mirror (630 DCXR, Chroma) and two
 bandpass filters (582/75 and 679/41 BrightLine series, Semrock). The
 excitation intensity was set to ~ 1 kW/cm<sup>2</sup> for both
 light sources. Each color was measured for 20 min at 10 Hz (100 ms
 exposure time) in the total internal reflection fluorescence (TIRF)
 illumination mode if not stated otherwise. Pixel size for data analysis
 was measured to 128 nm (red camera) and 132 nm (green camera). To ensure
 the alignment of both detection channels, a registration sample exhibiting
 identical patterns across the spectral ranges of both acquisition channels
 was obtained. For this purpose, fluorescent microsphere samples
 (TetraSpeck, Invitrogen, #T14792) with diameters of 0.2 µm were employed.
 The microspheres were positioned on the sample holder and it was verified
 that both cameras were physically aligned with a precision of one pixel.
 The excitation intensity was adjusted so that the fluorescence intensity
 was approximately equal across both channels. Data from both channels was
 recorded simultaneously, and the acquisition was repeated in various areas
 to achieve a high density of alignment markers in the reconstructed image
 of all microsphere recordings. Afterwards, the two-color samples were
 imaged and the corresponding imaging buffer, containing 3.5 nM of dye
 coupled imager strands respectively, were applied to the sample prior
 measurements. <strong>Data analysis for 2D
 TDI-DNA-PAINT. </strong>TDI-DNA-PAINT data for microtubules and
 DNA-origami were analyzed using the MATLAB-based Super-resolution
 Microscopy Analysis Platform (SMAP)<strong>
 </strong><em>(43)</em>.<strong>
 </strong>Using SMAP,<strong> </strong>data analysis was
 done by following the steps: loading of camera images, filtering,
 background estimation, finding of candidate molecules, fitting, and saving
 of the results. First, we loaded the raw blinking movies into the SMAP
 GUI, which was followed by manually setting up the camera parameters.
 Next, under the <em>Localize\Peak Finder </em>tab, we set the
 parameters for the initial estimation of single molecule positions, such
 as filtering cutoff with intensity values. To ensure selecting the
 brightest localizations, we select a higher cutoff value. After previewing
 an exemplary frame, we chose the Gaussian 2D fitting tab <em>PSF
 free</em> for localizing the full image set. The analysis returns
 coordinates such as x, y and z, number of photons per localization,
 background values, localization precisions, frame in which localization
 was found etc. Grouping (merging) of localizations persistent over several
 frames is performed after fitting. We can manually set the following
 grouping parameters dX (maximum distance two localizations can be apart)
 and dT (maximum number of dark frames between localizations). We grouped
 the localizations by setting dT = 1, and limiting the localization
 precision range from 0-15 nm, which filters out localizations with worse
 precision. On applying these grouping parameters, SMAP calculates the
 maximum distance from the localization precision of the two candidate
 localizations. That means that localizations are only grouped if it is
 likely they stem from the same event. We also quantified the background
 signal from different concentrations of Ox2-P3/R4 probes from 100 pM to 10
 nM, and 5 nM appeared to be the most optimal concentration considering it
 had very similar background as compared to 100 pM Ox1-P3 (fig. S3A). We
 also tested if the attachment of two fluorophores to the imager P3
 introduced any change to binding time (on-time). Figure S3C depict the
 on-time<em> </em>comparison between Ox1-P3, Ox2-P3, and
 Ox2-R4. Figures S3B and S3D-S3G presents photon yields, localization
 precision, background signal, and detected origami structures from
 TDI-DNA-PAINT experiments with Ox2-R4. The reported distances from origami
 experiments were from <em>N </em>= 33 values. For CD20
 TDI-DNA-PAINT datasets, analysis was performed following the same workflow
 as described and reconstructed images from 10 min acquisition is
 illustrated in Fig. 3B and figs. S8-S11.
 <strong>Data analysis for two-color TDI-DNA-PAINT.
 </strong>For the reconstruction of super-resolved two-color images,
 rapidSTORM3.3 was utilized. To align the two cameras employed, images of
 the recorded microsphere samples were reconstructed. A registration matrix
 was generated using the ImageJ plugin bUnwarpJ. The corresponding
 microsphere images were loaded, and 10 to 20 landmarks were identified
 using different microspheres in both images. Subsequently, the
 transformation matrix was converted into a raw transformation matrix. For
 the reconstruction of super-resolved microtubules and clathrin samples,
 the same settings used in the microsphere reconstruction were applied. The
 final reconstructed images for each channel were aligned using the ImageJ
 plugin bUnwarpJ along with the previously created transformation matrix.
 Finally, the aligned images were overlaid using ImageJ (v.
 2.3.0/1.54j). <strong>Whole-cell lattice light
 sheet (LLS) TDI-DNA-PAINT imaging. </strong> The workflow mostly
 recapitulates the description from M. K. Iwanski, E. A. Katrukha, L.
 Kapitein, <em>Methods Mol. Biol. </em>2694, 151-174
 (2024). <strong>Live-cell two-color LLS imaging
 of actin and CD20. </strong>Dual-color LLS imaging of Raji cells was
 carried out using the Zeiss lattice light-sheet 7. Live-cell incubation
 with 6% CO<sub>2 </sub>and 37 ˚C was maintained using the
 inbuilt stage top Incubator (ibidi, #12722). Actin was stained with the
 live-cell F-actin dye SPY555-FastAct™ (Spirochrome AG, #SC205) as
 described previously. RTX 5 μg/mL (Fig. 4A and fig. S14) or 10 μg/mL (fig.
 S15)), 2H7 5 μg/mL (Fig. 4C and fig. S16) or 10 μg/mL (fig. S17) or 20
 μg/mL (fig. S18)), OFA 5 μg/mL (Fig. 5A and fig. S21) or 10 μg/mL (Fig. 5B
 and fig. S22-S23), OBZ 5 μg/mL (Fig. 5C and fig. S24) or 10 μg/mL (Fig. 5D
 and fig. S25) or 20 μg/mL (Fig. 5E and fig. S26-S27), and were added to
 the cells in the respective sample chamber right before the start of
 experiment. Fluorophores were excited with “Sinc3 30 × 1000” light sheets
 for wavelengths (λ<sub>exc</sub>) 561 nm (SPY555-FastAct™) and
 640 nm (RTX-AF647/2H7-AF647/OBZ-ATTO643/OFA-ATTO643) via 13.3×/ 0.4 N.A.
 excitation objective lens. After focusing on the sample, imaging
 parameters such as correction for the X and Y tilt of the coverslip,
 “Focus Sheet” and “Focus Waist” were adjusted. The FOV in focus was
 scanned at 0.2 μm <em>y</em> stage position displacement in
 skewed dimension for a total range of 100 μm. Experiment was started right
 after adding RTX/2H7 to the chamber containing Raji cells and continued
 for 40 min with a sample scan repeating at 1 min interval. Emission light
 was collected through 44.83× /1.0 N.A. detection objective lens, followed
 by splitting to two cameras (ORCA-Fusion canal sCMOS (Hamamtsu Photonics),
 final pixel size 145 nm) with the aid of a 640 long-pass filter.
 Additionally, emission band-pass filters (576-617 and 656-750 nm) were
 also used before the respective cameras. Image analysis was performed
 using ZEN 3.9 blue software using the analysis method “Lattice light
 sheet”: with this protocol, acquired datasets were first deconvolved with
 a software in-built ‘Constrained Iterative’ deconvolution algorithm (6
 iterations), followed by deskewing the image datasets, and finally rotated
 to coverslip coordinates. Dual color images for RTX or 2H7 or OBZ or OFA
 (magenta) and actin (yellow) from these measurements are shown in figs.
 S14-S18 and S21-S25 with the same intensity gray value scaling for the
 respective channel across images for a final comparison of fluorescence
 signals between RTX and 2H7 and actin in different conditions tested.
 Movies S26-S35 depict Raji cell dynamics for 30 min at different RTX,2H7,
 OBZ, and OFA concentrations. <strong>Two-color
 LLS imaging of CD45 and CD20</strong>. Raji cells were labeled with
 5 µg/mL of either anti-CD20 RTX-AF647 or 2H7-AF647 and anti-CD45
 HI30-CF568 and fixed after RTX/2H7 incubation following the protocol
 described earlier. Two-color LLS imaging was performed using the
 commercial Zeiss lattice light-sheet 7 setup with the same settings as
 described in the previous section. Dual color Raji cell volumes (CD20
 (magenta) and CD45 (green)) shown in Fig. 4B and 4D and figs. S19-20
 demonstrates RTX accumulation in a polarized manner and its localization
 in membrane protrusions, corroborating our findings from live-cell LLS and
 LLS-TDI-DNA-PAINT measurements.
description: The provided .zip folders contain the data and plot variable values,
 arranged figure-wise. A. Ghosh*†, M. Meub†, D. A. Helmerich, J. Weingart,
 P. Eiring, T. Nerreter, K. M. Kortüm, S. Doose, and M. Sauer*. Decoding
 the molecular interplay of endogenous CD20 and therapeutic antibodies with
 fast volumetric nanoscopy Comments and requests should be addressed to
 Markus Sauer and Arindam Ghosh:
 [m.sauer@uni-wuerzburg.de](mailto:m.sauer@uni-wuerzburg.de);
 [arindam.ghosh@uni-wuerzburg.de](mailto:arindam.ghosh@uni-wuerzburg.de).
 All material is free of use, but we would appreciate being told, and this
 dataset and the paper cited where applicable. **Fig.1B-D** contains loc.
 files of 2D-TDI-DNA-PAINT imaging of cellular microtubules that are
 generated in the SMAP analysis platform (Jonas Ries lab) and the
 variables/columns follow exactly the SMAP guidelines. In particular, the
 provided .mat files contain localization attributes such as coordinates x,
 y, and z, the number of photons per localization, background values,
 localization precisions, frames in which localization was found, etc. Any
 user can open these files in SMAP (free to install and has a MATLAB-free
 version). 1E-G are plot variables with self-explanatory naming as .mat
 files.  For example, x-axis variables are stored in array 'x'
 and the same for y-axis variables as 'y'. 1H-I are loc. files
 .txt from 2-color TDI-DNA-PAINT imaging. **Fig. 2** contains a
 localization .csv file from whole cell LLS-TDI-DNA-PAINT imaging of
 cellular microtubule networks using TDI imager R4. The columns in the .csv
 files containing parameters such as coordinates x, y, and z, number of
 photons per localization, background values, and localization precisions,
 are intuitive to any non-specialist. Also, a .mat file containing
 calculated loc. precision values are provided. **Fig. 3B** contains
 localization .mat files from 2D TDI-DNA-PAINT experiments on CD20 labeled
 with Rituximab (RTX), 2H7, Ofatumumab (OFA), and Obinutuzumab (OBZ). These
 localization files are generated in SMAP analysis platform (Jonas Ries
 lab) and the variables/columns follow exactly as the SMAP guidelines. In
 particular, the provided .mat files contain localization attributes such
 as coordinates x, y, and z, number of photons per localization, background
 values, localization precisions, frame in which localization was found,
 etc. Any user can open these files in SMAP (free to install and has a
 MATLAB-free version). Any user can open these files in SMAP (free to
 install and has a MATLAB-free version). Fig. 3C-F contains localization
 files .csv from whole cell LLS-TDI-DNA-PAINT imaging of CD20 labeled with
 RTX, 2H7, OFA and OBZ. The columns in the .csv files are intuitive to any
 non-specialist containing coordinates x, y, and z, number of photons per
 localization, background values, and localization precisions, etc. Fig. 3G
 has the .mat file with localization density values calculated for each
 antibody sample. **Fig. 4** contains deconvolved and deskewed lattice
 light-sheet (LLS).czi files of the images from two-color live-cell imaging
 of CD20-RTX (5 μg/mL) and actin, CD20-2H7 (5 μg/mL) and actin, fixed cell
 two color images of CD20-RTX and CD45, and CD20-2H7 and CD45. All files
 were generated using Zeiss LLS microscope's Zen Blue software. Can be
 opened in Fiji/ImageJ. **Fig. 5A-E** contains deconvolved and deskewed
 lattice light-sheet (LLS).czi files of the images of two-color live-cell
 imaging of CD20-OFA (5 and 10 μg/mL) and actin, CD20-OBZ (5, 10, and 20
 μg/mL) and actin generated using Zeiss LLS microscope's Zen Blue
 software. Can be opened in Fiji/ImageJ. Fig. 5F contains self-explanatory
 plot variables (protrusion lengths) as a .mat file. **Fig. S1** contains
 absorbance spectra values of Ox2-P3 and Ox2-R4 TDI imagers as .mat files.
 **Fig. S2** contains fluorescence spectra values of TDI imagers labeled
 with four different dyes as .mat files. **Fig. S3** contains fluorescence
 spectra of Ox2-R4 and FCS analyzed data as .mat files. **Fig. S4**
 contains plot variables as .mat files for panels A,B,C,D,F,G and intensity
 grey values for panel E as a .csv file. **Fig. S5** contains localization
 file .txt from two-color 2D TDI-DNA-PAINT imaging of microtubules and
 clathrin-coated pits. The files can be opened through ImageJ plugin
 Thunderstorm and columns include parameters such as coordinates x, y, and
 z, number of photons per localization, background values, and localization
 precisions, etc. **Fig. S7** contains localization file .csv from whole
 cell LLS-TDI-DNA-PAINT imaging of cellular microtubule networks using TDI
 imager P3. The columns in the .csv files are intuitive to a non-specialist
 in the field. **Fig. S8** contains localization .mat files from 2D
 TDI-DNA-PAINT experiments on CD20-RTX. These loc. files are generated in
 SMAP analysis platform (Jonas Ries lab) and the variables/columns follow
 exactly as the SMAP guidelines. In particular, the provided .mat files
 contain localization attributes such as coordinates x, y, and z, number of
 photons per localization, background values, localization precisions,
 frame in which localization was found, etc. Any user can open these files
 in SMAP (free to install and has a MATLAB-free version). The folder also
 contains .txt files containing number of localizations plotted in S8C.
 **Fig. S9** contains localization .mat files from 2D TDI-DNA-PAINT
 experiments on CD20-2H7. These loc. files are generated in SMAP analysis
 platform (Jonas Ries lab) and the variables/columns follow exactly as the
 SMAP guidelines. In particular, the provided .mat files contain
 localization attributes such as coordinates x, y, and z, number of photons
 per localization, background values, localization precisions, frame in
 which localization was found, etc. Any user can open these files in SMAP
 (free to install and has a MATLAB-free version). The folder also contains
 .txt files containing number of localizations plotted in S9C. **Fig. S10**
 contains localization .mat files from 2D TDI-DNA-PAINT experiments on
 CD20-OFA. These loc. files are generated in SMAP analysis platform (Jonas
 Ries lab) and the variables/columns follow exactly as the SMAP
 guidelines.  In particular, the provided .mat files contain localization
 attributes such as coordinates x, y, and z, number of photons per
 localization, background values, localization precisions, frame in which
 localization was found, etc. Any user can open these files in SMAP (free
 to install and has a MATLAB-free version). **Fig. S11** contains
 localization .mat files from 2D TDI-DNA-PAINT experiments on CD20-OBZ.
 These loc. files are generated in SMAP analysis platform (Jonas Ries lab)
 and the variables/columns follow exactly as the SMAP guidelines. In
 particular, the provided .mat files contain localization attributes such
 as coordinates x, y, and z, number of photons per localization, background
 values, localization precisions, frame in which localization was found,
 etc. Any user can open these files in SMAP (free to install and has a
 MATLAB-free version). **Fig. S12** contains localization files .csv and
 reconstructed .tiff files from whole cell LLS-TDI-DNA-PAINT imaging of
 CD20 labeled with RTX and 2H7. The columns in the .csv files are intuitive
 to any non-specialist. MATLAB files provided contain loc. precision values
 plotted in S12E. **Fig. S13** contains localization files .csv and
 reconstructed .tiff files from whole cell LLS-TDI-DNA-PAINT imaging of
 CD20 labeled with OFA and OBZ. The columns in the .csv files are intuitive
 to a non-specialist in the field. **Fig. S14** contains deconvolved and
 deskewed lattice light-sheet (LLS).czi files of the images of two-color
 live-cell imaging of CD20-RTX (5 μg/mL) and actin generated by Zeiss LLS
 microscope's Zen Blue software. Can be opened in Fiji/ImageJ. **Fig.
 S15** contains deconvolved and deskewed lattice light-sheet (LLS).czi
 files of the images of two-color live-cell imaging of CD20-RTX (10 μg/mL)
 and actin generated by Zeiss LLS microscope's Zen Blue software. Can
 be opened in Fiji/ImageJ. **Fig. S16** contains deconvolved and deskewed
 lattice light-sheet (LLS).czi files of the images of two-color live-cell
 imaging of CD20-2H7 (5 μg/mL) and actin generated by Zeiss LLS
 microscope's Zen Blue software. Can be opened in Fiji/ImageJ. **Fig.
 S17** contains deconvolved and deskewed lattice light-sheet (LLS).czi
 files of the images of two-color live-cell imaging of CD20-2H7 (10 μg/mL)
 and actin generated by Zeiss LLS microscope's Zen Blue software. Can
 be opened in Fiji/ImageJ. **Fig. S18** contains deconvolved and deskewed
 lattice light-sheet (LLS).czi files of the images of two-color live-cell
 imaging of CD20-2H7 (20 μg/mL) and actin generated by Zeiss LLS
 microscope's Zen Blue software. Can be opened in Fiji/ImageJ. **Fig.
 S19_20** contains deconvolved and deskewed lattice light-sheet (LLS).czi
 files (large and small field of views) of the images of two-color fixed
 cell imaging of CD20-RTX (5 μg/mL) and CD45 generated by Zeiss LLS
 microscope's Zen Blue software. Can be opened in Fiji/ImageJ. **Fig.
 S21** contains deconvolved and deskewed lattice light-sheet (LLS).czi
 files of the images of two-color live-cell imaging of CD20-OFA (5 μg/mL)
 and actin generated by Zeiss LLS microscope's Zen Blue software. Can
 be opened in Fiji/ImageJ. **Fig. S22** contains deconvolved and deskewed
 lattice light-sheet (LLS).czi files of the images of two-color live-cell
 imaging of CD20-OFA (10 μg/mL) and actin generated by Zeiss LLS
 microscope's Zen Blue software. Can be opened in Fiji/ImageJ. **Fig.
 S24** contains deconvolved and deskewed lattice light-sheet (LLS).czi
 files of the images of two-color live-cell imaging of CD20-OBZ (5 μg/mL)
 and actin generated by Zeiss LLS microscope's Zen Blue software. Can
 be opened in Fiji/ImageJ. **Fig. S25** contains deconvolved and deskewed
 lattice light-sheet (LLS).czi files of the images of two-color live-cell
 imaging of CD20-OBZ (10 μg/mL) and actin generated by Zeiss LLS
 microscope's Zen Blue software. Can be opened in Fiji/ImageJ. **Fig.
 S26** contains deconvolved and deskewed lattice light-sheet (LLS).czi
 files of the images of two-color live-cell imaging of CD20-OBZ (20 μg/mL)
 and actin generated by Zeiss LLS microscope's Zen Blue software. Can
 be opened in Fiji/ImageJ.