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

Summary

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Abstract

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

Introduction

A variety of super-resolution microscopy techniques have been developed to overcome the diffraction limit of light microscopy (~200 nm). Among these is a category of techniques called single molecule localization microscopy (SMLM) which includes photo-activation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). SMLM techniques share in the use of fluorophores that can be switched between on (fluorescent) and off (dark/photo-switched) states, allowing sequential localization of fluorescence from single molecules^1,2,3.

Protocol

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in this protocol are toxic and carcinogenic. Please use all appropriate safety practices when performing the protocol including the use of engineering controls (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).

1. Multiplexed Imaging of Activated T Cells

1. Directly conjugate antibodies with Alexa-647 (A647) dye using the Alexa 647 antibody labeling kit. Follow the labeling protocol provided by the manufacturer.
   NOTE: Dialysis of antibo....

Results

The sequential elution and staining method was used to produce the multiplexed madSTORM image of microclusters and other structures in an activated Jurkat T cell (Figure 1, check figure alignments). Each pseudo-colored image represents one round of madSTORM imaging acquired in steps 1.1.1 to 1.3.13. As described previously¹⁰, the field of view should be monitored for residual signal from previous, non-eluted antibody to avoid crosstalk.......

Discussion

The sequential multiplexing, drift correction, and alignment procedures in madSTORM allow precise, highly multiplexed visualization of heterogenous structures in cells.¹⁰ In addition, madSTORM avoids the limitations of multi-color STORM such as chromatic aberration and sub-optimal photoswitching/emission properties of non-far red dyes^9,12. As the elution step significantly reduces steric interference from sequentially bound antibodies, mad.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8 well coverslip chamber	Lab-tek	155409
0.1% Poly-L-Lysine solution	Sigma-Aldrich	P8920
NV-100 nm Fluorescent Nano-diamond	Adamas	Red FND
anti-CD3ε antibody	BD Biosciences	555329
Bovine serum albumin, Fraction V	KSE Scientific	98-100P
Triton-X	Sigma-Aldrich	T9284
10% Paraformaldehyde	EMS	15712	Carcinogen
Gelatin from fresh water fish skin	Sigma-Aldrich	G7041
Alexa 647 antibody labeling kit	Thermo Fisher	A20186
Magnesium Chloride hexahydrate	Sigma-Aldrich	138924
PIPES	Sigma-Aldrich	P6757
Tween-20	Fisher Scientific	BP337-500
2-Mercaptoethanol	Sigma-Aldrich	M7154	Highly toxic, air sensitive
Cysteamine	Sigma-Aldrich	30070	Highly toxic
Cyclooctatetraene 98%	Sigma-Aldrich	138924	Highly toxic, air sensitive
10x PBS	KD Medical	RGF-3210
10x TBS	KD Medical	RGF-3385