Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Single molecule localization microscopy (SMLM) faces challenges in achieving high localization precision and multiplexing ability for imaging complex cellular structures.
• Existing methods struggle to accurately visualize the spatial organization of multiple proteins within cellular compartments.

Purpose of the Study:

• To improve localization precision in SMLM using fluorescent nano-diamond fiducial markers.
• To develop and demonstrate a novel multiplexing strategy, termed madSTORM, for simultaneous visualization of multiple molecular targets within a single cell.

Main Methods:

• Utilized fluorescent nano-diamond fiducial markers for drift correction and alignment in SMLM.
• Developed madSTORM, a multiplexing technique involving sequential binding and elution of fluorescent antibodies to target multiple molecules.
• Applied madSTORM to visualize components of T cell receptor microclusters in activated T cells.

Main Results:

• Achieved high precision in SMLM imaging through refined drift correction and alignment procedures.
• Successfully demonstrated the madSTORM strategy for multiplexed imaging of protein components within T cell receptor microclusters.
• Visualized the spatial organization of different proteins within a key immune cell signaling structure.

Conclusions:

• Fluorescent nano-diamond fiducial markers significantly enhance localization precision in SMLM.
• The madSTORM technique offers a versatile approach for multiplexed imaging of multi-protein structures in cell biology.
• This methodology advances the ability to study complex molecular architectures in cellular environments.