Eight years of single-molecule localization microscopy

Abstract

Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

Fig. 1

Live-cell dSTORM with SNAP tags. a Fluorescence image of histone H2B proteins in a COS-7 cell stained with SNAP-Cell TMR-Star (1 μM). Scale bar 5 μm. b Fluorescence image of the same cell but with 532 nm excitation of ~1 kW cm⁻², which induced photoswitching. c dSTORM image reconstructed from 10,000 images (acquired at 50 Hz). Adapted from (Klein et al. 2011), with permission

Fig. 2

a Nucleus of a Xenopus laevis A6 cell stained against the nuclear pore complex protein gp210 with pale white bar indicating the area where the x–z-cross section b is taken; c and d show the respective x–y- and y–z-views of the distal appendage protein CEP152 of centrioles from a U2OS cell; e represents another pair of centrioles in a COS-7 cell. All stainings were performed with Alexa Fluor 647. Scale bar a, b 1 μm; c, d 200 nm; e 500 nm; color-code (blue to red) a, b 0–4.6 μm; c, d 0–400 nm

Fig. 3

Three-dimensional PALM imaging of a vimentin network. a xy (top) and xz (bottom) maximum-intensity projections of PA-mCherry1–vimentin. About 1 million unlinked localizations was rendered in each view. Insets show further magnification of white rectangles in xy (lines in xz) maximum-intensity projection, highlighting individual vimentin fibrils in 60-nm-thick z-slices (localizations are linked). Arrow region of fibril with apparent width <100 nm. b Axial extent of vimentin network with z-location indicated as a color map. For clarity, localizations corresponding to 0–1.5 μm (top) and 1.5–3 μm (bottom) are shown separately. Arrowheads in a and b indicate a fibril that persists over >2 μm axially. Only linked localizations with correlation strength >0.4 are shown. Histogram bin sizes are 60 nm for all subfigures. Scale bars 3 μm (a), 600 nm (insets), 3 μm (b). Reproduced from York et al. (2011), with permission