Super-resolution fluorescence imaging with single molecules

Abstract

The ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence enables an emergent class of super-resolution microscopy methods which have overcome the longstanding diffraction barrier for far-field light-focusing optics. Achieving spatial resolutions of 20-40nm or better in both fixed and living cells, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. Briefly covering the basic principles, this short review then summarizes key recent developments and application examples of two-dimensional and three-dimensional (3D) multi-color techniques and faster time-lapse schemes. The prospects for quantitative imaging - in terms of improved ability to correct for dipole-emission-induced systematic localization errors and to provide accurate counts of molecular copy numbers within nanoscale cellular domains - are discussed.

Figure 1

Principles of super-resolution single-molecule active control microscopy. (a) A hypothetical arrangement of fluorescent molecules, i.e. a labeled “super-structure” (here: outline of “La Paloma de la Paz” (The Dove of Peace) by P. Picasso, 1961). (b) In conventional fluorescence microscopy, all molecules emit simultaneously, so their diffraction-limited images overlap on the detector (camera) and information about the underlying structure is irretrievably lost. (c) Addition of on-off control, toggling any one single-molecule emitter between a dark and a fluorescent state. (d) If individual sparse subsets of single molecules that are spatially separated further than the diffraction limit are made to emit, their positions may be extracted in a time-sequential manner by finding the center position of a mathematical fit of the single-molecule images. (e) From the list of localized molecules, a super-resolution reconstruction is assembled in a post-processing step. Note that if the majority of molecules is detected at least once, the resolution is then governed by the fidelity of the localization estimate of individual localizations. This precision is shown by the blue circles which, for reasonable signal-to-background of single-molecule detection, are dramatically smaller than the extent of the diffraction-limited image given by the microscope PSF. Scale bar: 250 nm. (f) The pixelated images of single-molecule emissions in a 2D imaging experiment are typically (g) fit by a Gaussian function with variable center coordinates (x,y) to extract (h) single-molecule position estimates. (i) Illustration of the inherent trade-off between spatial and temporal resolution when imaging a dynamic process: Cartoon view based on a general membrane fusion scenario, e.g. SNARE-mediated [87], evolving from a membrane stalk between a vesicle (top membrane) and the plasma membrane (bottom membrane) to a resulting fusion pore. If temporal resolution is prioritized, two or more reconstructions can be obtained, however a lower and possibly insufficient number of position samplings in the reconstructions – possibly also at worse localization precision – leave details unresolved. By contrast, collecting many positions while the structure is changing leads to time-averaging over the acquisition, and a similar loss of information.

Figure 2

Examples of single-molecule-based super-resolution imaging in biological applications. (a) The nucleoid-associated protein HU imaged at different stages in the cell cycle of asymmetrically dividing C. crescentus bacterial cells, by light-induced blinking of enhanced yellow fluorescent protein fusions. (b) A spindle-like apparatus of ParA is part of the asymmetric division machinery in C. crescentus bacterial cells. (c) Nuclear pore complexes (gp210 proteins) with eight-fold symmetrical subunits in isolated Xenopus laevis oocyte nuclear envelopes. (d) Cytosolic nanoscale fibrillar aggregates of mutant huntingtin exon 1 proteins inside intact neuronal model cells (PC12m). IB: inclusion body. Nu: nucleus (e) tdEos-tagged focal adhesion molecules (paxillin) imaged in 2D at 55-s time resolution in a live CHO cell. (f) Endoplasmic reticulum (ER) dynamics in live BS-C-1 cells. (left) A time-series of 10-sec STORM snapshots. Blue arrowheads: Tips of extending tubules. (middle) A composite image containing all these snapshots with each localization colored by its time of appearance according to the shown color map. (right) Distribution of the widths of ER tubules. Green bars: Newly extended tubules. Red bars: Old tubules that had already existed for at least 2 min. (g) Voltage-gated ion channels recognized by fluorescently-labeled saxitoxin ligands, imaged at 6 seconds / reconstruction. Spines are constantly extending and retracting from the extended neuritic process in the neuronal model cell PC12. Examples reproduced with permission from [35] (a), [38] (b), [40] (c), [44] (d), [31] (e), [51] (f), [46] (g).

Figure 3

Three-dimensional (3D) super-resolution fluorescence imaging. (a) To extract the location of individual single-molecule emitters with high precision in all three spatial dimensions, the symmetry of the standard microscope PSF (i) must be broken and the PSF re-designed to encode information in the z (axial) direction by a well-defined shape change. Widely adopted schemes include: (ii) an astigmatic PSF, where the slowly changing ellipticity of the single-molecule image can be calibrated to provide a z estimate. (iii) the double-helix (DH) PSF features two well-defined spots revolving around a common center as a function of z. (iv) cork screw PSF (one revolving spot), (v) bi-plane methods assess the relative detected brightness of images formed in two shifted image planes. (vi) 4Pi axial localization methods rely on interferometric detection and two matched objective lenses to collect fluorescence from both sides of the sample. (b) Microtubules in mammalian cells extending over a large axial range can be readily imaged with the DH-PSF. (c) Visualization of the cytoskeletal protein filament of CreS in pre-divisional C. crescentus bacterial cells, jointly with the cell surface by the PAINT approach. (d) Quantitative co-localization of a further protein, PopZ (shown in red). The number density of PopZ in the polar nano-domains was demonstrated to be constant between cells. Both (c) and (d) recorded by DH-PSF microscopy. (e) Cell-wide arrangement of F-actin by dual-objective astigmatic imaging. Examples reproduced with permission from [32] (b), [88] (c), [89] (d), [33] (e).