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

Abstract

A highly multiplexed superresolution imaging strategy with single-molecule accuracy enabled by fluorescent nanodiamonds called madSTORM affords the ability to define spatial relationships among constituent molecules within structures. It makes it possible to probe the molecular topology of complex signaling cascades and other heterogeneous networks.

DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging

“…Hence, orthogonal imager strands can be used to sequentially visualize multiple targets of interest. This so-called Exchange-PAINT 15 approach in principle enables the spectrally-unlimited multiplexed super-resolution imaging of potentially hundreds of target molecules in the same sample, in a simpler and more straightforward fashion than other multiplexing approaches, 18–22 such as those based on sequential immunostaining, imaging, and dye bleaching or inactivation.…”

Differential nanoscale organisation of LFA-1 modulates T cell migration

“…T cells were first tracked during migration. Cells were fixed, and their positions were recorded, and then multiplexed antibody madSTORM (Yi et al 2016b;) was used to achieve multicolour imaging. 3 colour pointillist maps were analysed channel per channel with Bayesian cluster analysis (Griffié et al 2016), and colocalization was analysed (Rossy et al 2014).…”

“…As nanoclusters resemble nascent adhesions in non-leukocytes, but occur throughout the cell, we next investigated how they are regulated in T cells. We tested whether the lack of PTPN22 causes changes in the colocalization of pFAK with LFA-1 and a second set of signalling intermediates: the Src family kinases, using multi-colour SMLM microscopy (Yi et al 2016a;. Wild type or PTPN22 deficient primary murine T cell blasts were plated on ICAM-1, allowed to migrate and tracked by live-cell imaging.…”

“…Wild type or PTPN22 deficient primary murine T cell blasts were plated on ICAM-1, allowed to migrate and tracked by live-cell imaging. They were then fixed, stained and imaged using madSTORM (Yi et al 2016b) permitting the sequential imaging of pY397FAK, pY416Src and LFA-1. Supplementary Figure 3 confirmed that PTPN22 deficient cells were faster than PTPN22 proficient cells.…”

Estimation of microscope drift using fluorescent nanodiamonds as fiducial markers

“…FNDs are biocompatible, photostable with a high quantum efficiency (QE ß0.7), have a broad excitation spectra (ß450-580 nm) and have a broad emission spectra (ß640-700 nm) (Yu et al, 2005;Jelezko & Wrachtrup, 2006;Balasubramanian et al, 2008;Xing & Liming, 2009;Mochalin et al, 2012;Bumb et al, 2013) suitable for multicolour imaging. Recently, FNDs have been used as fiducial markers for single molecule tracking (Dittmore et al, 2016) and madSTORM super-resolution imaging (Yi et al, 2016). It should be noted that the reduced photostability due to oxygen or reducing thiols is not a concern for FNDs because fluorescent NV centres inside FNDs are isolated from the microenvironment.…”