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Decoding CD20: A New Era in Immunotherapy Imaging with Lattice Lightsheet 7 & Single-Molecule Localization

30 October 2025 6 min read byZEISS Microscopy
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Introduction

Revealing CD20-Antibody Dynamics in B Cells

Can we see how immunotherapies work at the molecular level, inside living cells? Until recently, imaging the real-time interplay between therapeutic antibodies and membrane receptors was limited by resolution, speed, and distortion – obscuring the mechanisms that determine treatment success.
At the Julius-Maximilians-University Wuerzburg (JMU), Germany, Prof. Dr. Markus Sauer and team have used ZEISS Lattice Lightsheet 7 with single-molecule localization to visualize CD20 receptor behavior on B cells. Their imaging reveals how antibody binding induces receptor clustering and cell polarization – offering new insights into immune activation.

Dynamic CD20 Clustering & Actin Dynamics in Raji B Cells Imaged with ZEISS Lattice Lightsheet 7

  • Two-color lattice light-sheet (LLS) imaging

    10 μg/mL OFA-stained CD20 (magenta) and actin (yellow) dynamics for 30 min after the start of the experiment.

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Advancing Personalized Immunotherapy Through Live-Cell Imaging of CD20 Clustering

This research opens new possibilities for personalized medicine. By enabling fast, volumetric live-cell imaging of antibody-receptor interactions, it supports the development of improved diagnostics, targeted therapies, and novel biomarkers.

These insights could accelerate antibody design and enhance treatment strategies for autoimmune diseases and cancer – with global relevance for clinical research and patient care. 

A middle-aged man with short gray hair and light stubble, wearing a blue shirt and dark jacket, looks at the camera with a neutral expression against a pale background.

We’re not just pushing resolution limits – we’re translating imaging into diagnostics and treatment.

Prof. Dr. Markus Sauer Professor for Biotechnology and Biophysics at the Julius-Maximilians-University Würzburg, Germany
A 3D microscopic image showing clusters of spherical cells highlighted in yellow and pink on a black background with grid lines.
A 3D microscopic image showing clusters of spherical cells highlighted in yellow and pink on a black background with grid lines.

Large volumetric imaging of the effect of monoclonal antibodies (mAbs) on B cells, conducted using two-color lattice light-sheet (LLS) imaging of CD20 and CD45 in Raji B cells. Raji B cells labeled with 5 μg/mL anti-CD20 RTX-AF647 (magenta) and anti-CD45 antibody-CF568 (yellow), incubated for 30 minutes at room temperature (RT) following RTX addition. Cells fixed post-incubation. Deconvolved LLS images clearly demonstrate the ‘polarized’ accumulation and clustering of CD20 in membrane protrusions, while CD45 is ubiquitously distributed throughout the cell membrane.

Large volumetric imaging of the effect of monoclonal antibodies (mAbs) on B cells, conducted using two-color lattice light-sheet (LLS) imaging of CD20 and CD45 in Raji B cells. Raji B cells labeled with 5 μg/mL anti-CD20 RTX-AF647 (magenta) and anti-CD45 antibody-CF568 (yellow), incubated for 30 minutes at room temperature (RT) following RTX addition. Cells fixed post-incubation. Deconvolved LLS images clearly demonstrate the ‘polarized’ accumulation and clustering of CD20 in membrane protrusions, while CD45 is ubiquitously distributed throughout the cell membrane.

A Translational Imaging Breakthrough

The team’s research focuses on single-molecule sensitive fluorescence detection and super-resolution microscopy. By combining TDI-DNA-PAINT with Lattice Lightsheet 7, they achieved fast, volumetric imaging of whole B cells – capturing the dynamic interplay between CD20 and therapeutic antibodies like Rituximab (RTX), Ofatumumab (OFA), and Obinutuzumab (OBZ).

This approach revealed that both type I and type II antibodies induce CD20 clustering and B cell polarization – challenging long-standing classifications and offering new perspectives on how immune responses are triggered.

Guided by the pursuit of molecular precision, the team at JMU focuses on single-molecule sensitive fluorescence detection and super-resolution microscopy – uncovering how cellular behavior unfolds at the nanoscale. 

Single-molecule imaging reveals the individuality of cells – it’s the foundation for personalized treatment.

Prof. Dr. Markus Sauer Professor for Biotechnology and Biophysics at the Julius-Maximilians-University Würzburg, Germany
Four 3D fluorescence microscopy images show cell clusters with green and magenta-stained structures, displayed within grid backgrounds and 5 μm scale bars.
Four 3D fluorescence microscopy images show cell clusters with green and magenta-stained structures, displayed within grid backgrounds and 5 μm scale bars.

Two-color LLS imaging of CD20 and CD45 in Raji B cells. (A) Raji B cells labeled with 5 μg/mL anti-CD20 RTX-AF647 (magenta), anti-CD45 antibody-CF568 (green), incubated for 30 minutes at room temperature (RT) following RTX addition. Cells fixed post-incubation. Deconvolved LLS images demonstrate ‘polarized’ CD20 clusters and their accumulation in microvilli, while CD45 is distributed throughout the cell membrane. (B) Raji cells labeled with 5 μg/mL anti-CD20 2H7-AF647 (magenta) demonstrate significantly reduced signal globally as compared to (A) due to lower binding affinity of 2H7 and reduced CD20 clustering and stabilization of microvilli unlike RTX.
Intensity gray value scaling was the same for CD20-RTX and CD20-2H7 channels, and the same for CD45 channels in (A) and (B).

Two-color LLS imaging of CD20 and CD45 in Raji B cells. (A) Raji B cells labeled with 5 μg/mL anti-CD20 RTX-AF647 (magenta), anti-CD45 antibody-CF568 (green), incubated for 30 minutes at room temperature (RT) following RTX addition. Cells fixed post-incubation. Deconvolved LLS images demonstrate ‘polarized’ CD20 clusters and their accumulation in microvilli, while CD45 is distributed throughout the cell membrane. (B) Raji cells labeled with 5 μg/mL anti-CD20 2H7-AF647 (magenta) demonstrate significantly reduced signal globally as compared to (A) due to lower binding affinity of 2H7 and reduced CD20 clustering and stabilization of microvilli unlike RTX.
Intensity gray value scaling was the same for CD20-RTX and CD20-2H7 channels, and the same for CD45 channels in (A) and (B).

Why Lattice Lightsheet 7?

ZEISS Lattice Lightsheet 7 was chosen for its intuitive design and compatibility with single-molecule localization techniques. While the team had access to a Betzig LLS system, they found ZEISS Lattice Lightsheet 7 to be more efficient and easier to use.

Its ability to image entire cells in 3D with minimal photobleaching enabled the team to visualize CD20 distributions at apical sites and cell-cell contact zones – areas previously difficult to access without surface-induced artifacts.

A middle-aged man with short gray hair wearing a dark polo shirt stands in front of a staircase with a curved railing.

Lattice Lightsheet 7 makes whole-cell imaging accessible – no complex alignments, just powerful results.

Prof. Dr. Markus Sauer Professor for Biotechnology and Biophysics at the Julius-Maximilians-University Würzburg, Germany
3D scatter plot with red/orange data points clustered in the center, labeled axes in micrometers: X (0–25), Y (0–15), and Z (0–10), on a black background.

3D LLS-TDI-DNA-PAINT volume rendering of CD20/mAb complexes in Raji cells labeled at room temperature (RT) with 5 μg/mL monoclonal antibody (mAb).

Engineering for Clarity

To overcome limitations in traditional DNA-PAINT, the team developed two-dye imager strands that form non-fluorescent dimers in the unbound state. This technique – an advanced version of DNA-PAINT single-molecule localization microscopy – reduces background noise and allows for higher probe concentrations, resulting in imaging speeds up to 15 times faster.

They also adopted the introduction of astigmatism with the system's inherent custom-engineered optics, diverting their intended use for aberration control to enable precise 3D localization. This solution was first introduced by the group of Lukas Kapitein, a leading cell biologist at Utrecht University known for pioneering lattice light-sheet motor-PAINT to study microtubule orientation in whole cells.

We minimized background and boosted speed – now we can image whole cells in hours, not days.

Prof. Dr. Markus Sauer Professor for Biotechnology and Biophysics at the Julius-Maximilians-University Würzburg, Germany

Implications for Immunotherapy

The ability to visualize CD20 clustering and B cell polarization in real time opens new doors for understanding how therapeutic antibodies activate immune responses. These insights could guide the design of next-generation immunotherapies and support more personalized treatment strategies.

The approach has already attracted attention from pharmaceutical companies exploring new antibody formats and precision diagnostics.

The future is molecular clarity – seeing every detail in 3D, in living cells.

Prof. Dr. Markus Sauer Professor for Biotechnology and Biophysics at the Julius-Maximilians-University Würzburg, Germany
A tablet displaying a scientific research article summary with text, diagrams, and graphs on the screen.

CD20 Imaging in Live B Cells: Sauer’s Team Pushes Molecular Boundaries

Learn how Lattice Lightsheet 7 is transforming single-molecule imaging in immunology:

Two women work in a lab; one operates a large machine on a table, while the other sits at a computer desk with multiple monitors.
Two women work in a lab; one operates a large machine on a table, while the other sits at a computer desk with multiple monitors.

Scientists develop advanced fluorescence imaging techniques – including dSTORM, TDI-DNA-PAINT, expansion microscopy, and photoswitching fingerprint analysis – to drive innovation in diagnostics and immunotherapy.

Scientists develop advanced fluorescence imaging techniques – including dSTORM, TDI-DNA-PAINT, expansion microscopy, and photoswitching fingerprint analysis – to drive innovation in diagnostics and immunotherapy.

Research Focus at JMU

At the Julius-Maximilians-University Würzburg (JMU), Prof. Dr. Markus Sauer leads a research group specializing in single-molecule fluorescence detection and super-resolution microscopy. With a strong foundation in dye chemistry and photophysics, the team develops advanced imaging techniques such as

  • dSTORM,
  • TDI-DNA-PAINT,
  • expansion microscopy, and
  • photoswitching fingerprint analysis.

Their work bridges fundamental biophysics with translational applications, aiming to improve diagnostics and immunotherapy – particularly through the molecular characterization of therapeutic antibodies and CAR-T cells.

As one of the first groups to publish in this field, they continue to push the boundaries of resolution and real-world impact – positioning JMU as one of the leaders in single-molecule biophysics.

LinkedIn profile of Prof. Dr. Markus Sauer
 

In Brief

  • To visualize CD20 receptor clustering in intact B cells, researchers use advanced volumetric imaging techniques that combine lattice light-sheet microscopy with high-resolution localization methods. This approach enables precise mapping of receptor distributions and cell polarization in three dimensions – even in live-cell conditions.

  • Tracking antibody-receptor interactions at the molecular level requires techniques that resolve individual molecules with nanometer precision. By integrating lattice light-sheet microscopy with localization-based imaging, researchers can observe how therapeutic antibodies bind, cluster, and activate receptors like CD20 in real time.

  • To reduce background and accelerate acquisition in high-resolution microscopy, researchers use optimized probe designs such as two-dye imager strands. These strands minimize fluorescence noise and allow higher probe concentrations, enabling faster and clearer imaging of cellular structures – especially in large or expanded samples.
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