Multitarget super-resolution microscopy with high-density labeling by exchangeable probes

Abstract

We have developed a multitarget super-resolution microscopy technique called image reconstruction by integrating exchangeable single-molecule localization (IRIS). IRIS uses protein fragment-based probes that directly associate with and dissociate from their targets over durations on the order of tens of milliseconds. By integrating single-molecule localization and sequential labeling, IRIS enables unprecedented labeling density along multiple cellular structures. IRIS can be used to discern the area-specific proximity between cytoskeletal components and focal adhesions within a single cell.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

“…As Y-FAST interconverts spontaneously and rapidly at the singlemolecule level between a dark (unbound) state and a bright (bound) state, it should behave as a blinking fluorophore (44). Fine-tuning of the exchange dynamics could give access to blinking rates adequate for Single-molecule Localization Microscopies (41,45,46) or Superresolution Optical Fluctuation Imaging (44,47).…”

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

“…As such, madSTORM can be viewed as a topology tool, mapping the locations of diverse molecules within structures and complex signaling networks at single-molecule accuracy. This is in contrast to the common application of SMLM in imaging the shapes and patterns of structures, which requires significantly higher signal density of probes than is possible using antibody-based SMLM methods (Kiuchi et al ., 2015; Legant et al ., 2016). For these reasons, signal density has been sacrificed to achieve the high localization precision needed for single-molecule accuracy.…”

“…dSTORM has been used for two purposes: visualizing structures at high resolution (requires high labeling density) and accurately locating single-molecule positions (requires high precision). Regarding the first purpose, a recent study showed that even at maximum labeling density of fluorescent antibody molecules, discontinuous patterns are observed along bundled actin filaments (Kiuchi et al ., 2015), making it difficult to reach nanoscale resolution as defined by the Nyquist criterion. Furthermore, a separate study showed that five times the labeling density as calculated by the Nyquist criterion is required to properly resolve nanostructures such as F-actin (Legant et al ., 2016).…”

“…Furthermore, the accumulation of fluorescent probes could lead to steric blocking of binding sites in the sample, preventing large-scale multiplexing and robust targeting of epitopes. To avoid such steric interference, a recent study achieved multiplexing using stochastic exchange of freely diffusing protein fragments (Kiuchi et al ., 2015). Although this method allows dense labeling of cellular structures, it requires extensive biochemical preparation to isolate peptide fragments, cannot locate single-molecule positions, and does not readily facilitate large-scale multiplexing using commercially available probes.…”

Quantification of fibrous spatial point patterns from single-molecule localization microscopy (SMLM) data

“…A recently developed SMLM method, IRIS (Kiuchi, et al, 2015), has demonstrated unparalleled labelling density across various cellular structures, thereby offering a considerable improvement to conventional probe methods. IRIS probes are small fluorescently labelled protein fragments which rapidly bind and unbind to their specific target structures.…”