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Updated: Mar 14, 2026

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

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

Published on: June 24, 2017

madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy.

Jason Yi¹, Asit Manna¹, Valarie A Barr¹

¹Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

Molecular Biology of the Cell

|October 7, 2016

Summary

Researchers developed madSTORM, a new method using fluorescent nanodiamonds, to precisely map multiple molecules in cellular structures. This advance improves localization accuracy and multiplexing for detailed molecular analysis.

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Last Updated: Mar 14, 2026

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

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Published on: October 28, 2018

Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Single-molecule localization microscopy (SMLM) faces limitations in localization accuracy and multiplexing capacity for studying heterogeneous cellular structures.
Precisely defining the spatial organization of molecules within cellular structures is crucial for understanding biological functions.

Purpose of the Study:

To enhance localization accuracy and multiplexing capabilities in SMLM for detailed cellular structure investigation.
To develop a novel method for accurate multi-epitope targeting and localization within complex biological systems.

Main Methods:

Utilized fluorescent nanodiamonds as fiducial markers to define and achieve high localization precision in dSTORM (direct Stochastic Optical Reconstruction Microscopy) imaging.
Introduced madSTORM, a multiplexing strategy employing sequential binding and elution of fluorescent antibodies for multi-target localization.
Applied madSTORM to activated T-cells to localize multiple epitopes, including those within the T-cell receptor complex.

Main Results:

Achieved single-molecule localization accuracy with an average precision of 2.6 nm and an alignment error of 2.0 nm.
Demonstrated successful multiplexing by localizing 25 epitopes in an activated T-cell, with 14 belonging to the T-cell receptor complex.
Reported minimal cross-talk (<0.01%) between targeted molecules, validating the efficacy of the madSTORM strategy.

Conclusions:

The developed madSTORM method significantly improves localization precision and multiplexing capacity in SMLM.
This technique enables the precise mapping of spatial relationships among constituent molecules within complex cellular structures.
madSTORM facilitates the study of molecular topology in signaling cascades and other heterogeneous biological networks.