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MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution
Contributed by Stefan W. Hell, April 30, 2018 (sent for review February 1, 2018; reviewed by Taekjip Ha and Benjamin Schuler)

Significance
Popular localization of single molecules through calculating the centroid of the diffraction pattern produced by molecular fluorescence on a camera is typically limited to spatiotemporal resolutions of >10 nm per >10 milliseconds. By requiring at least 10–100 times fewer detected photons and being free of bias due to molecular orientation, the localization concept called MINFLUX propels molecular tracking to the hitherto-unachievable regime of single-digit nanometer precision within substantially less than a millisecond. Our experiments herald the feasibility to detect molecular interactions and conformational changes at microsecond timescales.
Abstract
Compared with localization schemes solely based on evaluating patterns of molecular emission, the recently introduced single-molecule localization concept called MINFLUX and the fluorescence nanoscopies derived from it require up to orders of magnitude fewer emissions to attain single-digit nanometer resolution. Here, we demonstrate that the lower number of required fluorescence photons enables MINFLUX to detect molecular movements of a few nanometers at a temporal sampling of well below 1 millisecond. Using fluorophores attached to thermally fluctuating DNA strands as model systems, we demonstrate that measurement times as short as 400 microseconds suffice to localize fluorescent molecules with ∼2-nm precision. Such performance is out of reach for popular camera-based localization by centroid calculation of emission diffraction patterns. Since theoretical limits have not been reached, our results show that emerging MINFLUX nanoscopy bears great potential for dissecting the motions of individual (macro)molecules at hitherto-unattained combinations of spatial and temporal resolution.
Fluorescence nanoscopy methods such as those called stimulated emission depletion (STED)/reversible saturable optical linear fluorescence transitions (RESOLFT) microscopy (1, 2) and photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM) (3⇓–5) routinely achieve 20- to 50-nm spatial resolution (6⇓–8) with recording times down to tens of milliseconds, albeit with the requirements for fast recording and high spatial resolution difficult to reconcile. By synergistically combining the strengths of the two superresolution families, the recent MINFLUX concept is bound to solve this problem.
MINFLUX is based on the insight that, while both STED/RESOLFT and PALM/STORM modulate fluorescence emission to distinguish nearby molecules, they differ at establishing the molecular position (9). Whereas in STED/RESOLFT the position of the molecule is determined by interrogating the sample with a spatially controlled illumination pattern, for example, a doughnut or a standing wave, in PALM/STORM the molecular position is inferred from the pattern of fluorescence light rendered by a single molecule on a pixelated detector, such as a camera (10). Concretely, the molecule’s position is equated with the maximum of the fluorescence diffraction pattern, following backprojection into sample space. The precision of this camera-based “localization” ideally reaches
The need for large photon detection numbers N has spurred the development of related methods, such as confocalized tracking with multiple point detectors (16, 17), detection PSF engineering (18), Gaussian beam scanning schemes (i.e., orbital tracking) (19⇓⇓⇓⇓–24), and multiplexed excitation schemes (25⇓⇓–28). While all these methods provide specific advantages, none of them solved the need for large detection events N, keeping the photon emission rate as the limiting factor for the obtainable spatiotemporal resolution. As a result, current state-of-the-art single-molecule tracking performance has remained in the tens of nanometer per several tens of millisecond range.
Drawing on the basic rationale of the coordinate determination employed in STED/RESOLFT microscopy, MINFLUX has addressed these fundamental limitations (29). By localizing individual emitters with an excitation beam featuring an intensity minimum that is spatially precisely controlled, MINFLUX takes advantage of coordinate targeting for single-molecule localization. In a typical 2D MINFLUX implementation, the position of a molecule is obtained by placing the minimum of a doughnut-shaped excitation beam at a known set of spatial coordinates in the molecule’s proximity. These coordinates are within a range L in which the molecule is anticipated (Fig. 1). Probing the number of detected photons for each doughnut minimum coordinate yields the molecular position. It is the position at which the doughnut would produce minimal emission, if the excitation intensity minimum were targeted to it directly. As the intensity minimum is ideally a zero, it is the point at which emission is ideally absent. The precision of the position estimate increases with the square root of the total number of detected photons and, more importantly, by decreasing the range L. For small ranges L at which the intensity minimum is approximated by a quadratic function, the localization precision does not depend on any wavelength and, for the case of no background and perfect doughnut control, the precision
MINFLUX probes the position of an emitter with light distributions featuring intensity zeros that are targeted to defined coordinates in sample space. (A) Schematic of the MINFLUX setup used here. A laser beam is structured by a phase mask to obtain a doughnut-shaped excitation profile
Here, we demonstrate nanometer-precision tracking of a single emitter quickly fluctuating within a 20-nm range. We also show that, for attaining a certain precision in a predefined region of interest (ROI), there is an optimal distance L, depending on the signal-to-background ratio (SBR) and the ROI size. Establishing the optimal L allowed us to increase the spatiotemporal resolution beyond reported values, as demonstrated by tracking the stochastic movement of a labeled DNA construct with submillisecond resolution and ∼2-nm precision.
Results
Nanometer-scale MINFLUX tracking measurements were carried out with a custom-built laser-scanning fluorescence microscope featuring fast beam deflection and modulation (SI Appendix, Fig. S2A) that followed a previously reported design (29). The excitation beam is doughnut-shaped
Studying nanoscale movements using this stationary STC requires the molecules of interest to be placed proximal to the STC center, because this is where localization precision is highest. The precision deteriorates toward the STC periphery, depending on L (SI Appendix, Fig. S8): small values of L yield high precision in small regions, whereas large L values exhibit poorer precision over a larger area. Thus, this parameter needs to be adapted to the size of the ROI. As a cost function for optimizing L, we chose the average localization Cramér-Rao bound (CRB) over the edge of a circular ROI with diameter
This relationship indicates which L to choose for a given ROI. The MINFLUX CRB values inside this region for a total of
Because MINFLUX requires fewer detected photons, molecules can be localized at higher temporal resolution. For a detection count rate of
MINFLUX Performance Evaluation.
The experimental MINFLUX performance at high temporal resolution was characterized by repeatedly localizing individual immobilized ATTO 647N fluorophores within the ROI (Fig. 1C). Our measurements used
We first evaluated the experimental MINFLUX localization precision for temporal resolutions of 0.1–1 ms, attained by 1- to 10-fold binning of the measured count traces. For a b-fold binning, the detected photons from each of the four exposures of an STC cycle were added to those of the following
An example of the position estimates for a 300-ms trace extract is shown in Fig. 2C. Evaluation of the resulting localization precision σ using all 13,625 localizations of the trace yields
Fluorescence time traces of a single molecule and resulting localization precision at high temporal resolution using MINFLUX. (A) Diagram of the DNA origami construct with a single ATTO 647N fluorophore attached closely to a glass surface. Immobilization was achieved by complementarily pairing a ssDNA linked to an ATTO 647N molecule with a second ssDNA that is attached to a rectangular DNA origami (SI Appendix, Sample Preparation). (B) Histogram of 13,625 MINFLUX localizations of a sample with 1 × 1-nm binning. Time resolution,
Since optimized centering within the STC range is not possible for all emitters (SI Appendix, Data Acquisition), different positions within the ROI provide slightly different precision. However, throughout a ROI with dROI = 30 nm and for all temporal resolutions evaluated, the localization precision is improved over that provided by ideal camera localization (Fig. 1E). On the other hand, the measured localization precisions did not fully reach their CRB. The precision of
Nanometer-Scale MINFLUX Tracking.
To demonstrate its ability to record fast movements of target molecules with high spatiotemporal resolution, we applied MINFLUX to the detection of rapid movements of a custom-designed DNA origami sample (Fig. 3A). Two extended ssDNA attached to a modified Rothemund rectangle (31) were base paired with a DNA bridge strand. An ATTO 647N emitter was attached approximately at the center of the bridge, resulting in a maximal distance of about 10 nm from the DNA origami base (SI Appendix, DNA Origami Sample Preparation). This 1D diffuser (1DD bridge) can only move on a half-circle above the DNA origami by design, ideally yielding a 1D movement. For a maximal emitter displacement of 20 nm, the predicted optimal L value is
MINFLUX tracking of rapid movements of a custom-designed DNA origami. (A) Diagram of the DNA origami construct with a single ATTO 647N fluorophore attached at the center of the bridge (10 nm from the origami base). By design, the emitter can move on a half-circle above the origami and is thus ideally restricted to a 1D movement. (B) Histogram of 6,118 localizations of the sample in A with δt = 400-µs time resolution and a 1.5 × 1.5-nm binning. The predominant motion is along a single direction (
Like for the static DNA construct, a 2D histogram of the localizations (Fig. 3B) and photon counts (Fig. 3C, Upper) together with the trajectory trace excerpt (Fig. 3C, Lower) are shown for the 1DD (Movies S1 and S2). Again, acquisition was performed at 100 µs per STC cycle and the counts binned to
For a perfect 1D movement, the localization precision perpendicular to
As we measured the projection of a 3D movement on a 2D plane, the result resembles a translocation with two predominant positions on the principal axis. Fig. 3D shows a 14-ms excerpt containing 35 localizations in which the transitions between the predominant positions (marked by black circles) are clearly resolved. The distance between these positions is
Discussion and Conclusion
MINFLUX operates by targeting one or more intensity minima of an excitation light pattern to a defined set of positions. Matching the positions closely with that of the emitter reduces the number of fluorescence photons required for localization compared with conventional schemes that extract molecular positions just from emission patterns. Due to this conceptual advantage, MINFLUX has the power to attain higher spatiotemporal resolution, as demonstrated in this study.
As for virtually every fluorescence technique, the presence of background emission and optical aberrations (which affects in turn the contrast and absolute depth of the intensity minima) restrict the performance of MINFLUX, limiting how efficiently the collected photons can be used. Furthermore, the fluorophore saturation sets a bound for the achievable spatiotemporal resolution, and the emission blinking naturally limits the temporal resolution.
While the sub-10-nm scale can also be accessed through near-field interactions using a nanosized tip or Förster resonant energy transfer (FRET), a strength of MINFLUX is that it can investigate the nanometer scale from virtually any distance. Besides, unlike FRET, MINFLUX does not entail a photophysically constrained range of action (32) nor does it require multiple labels. Nevertheless, one can envisage combining MINFLUX with FRET to extract further information about molecules such as short distance interactions, rotations, and translations.
We further showed that in a practical MINFLUX implementation, the presence of background leads to an optimal spatial range L of the set of coordinates used for probing the molecular position. Reducing this range does not improve performance further. By the same token, optimizing our range L allowed us to reach spatial precisions
The advancement in spatiotemporal resolution was demonstrated through directly observing nanometer molecular movements over extended periods (of 5.9 s on average), yielding tens of thousands of localizations. The demonstrated performance makes biological applications of MINFLUX foreseeable, especially in a multicolor implementation. For example, MINFLUX should improve the observation of rotations and movements of molecular motors as well as of protein folding. The unique combination of localization precision and recording speed should also enhance our understanding of protein and lipid diffusion in cell membranes. In general, MINFLUX localization should trigger a number of single-molecule–based spatiotemporal investigations that have been so far precluded by the attainable fluorescence flux.
Materials and Methods
A description of the sample preparation, the optical setup, data acquisition, as well as data analysis including the emitter localization can be found in SI Appendix, Materials and Methods. In brief, MINFLUX was implemented in a custom-built confocal laser-scanning microscope with fast beam positioning and modulation capabilities. Electro-optical deflectors achieved rapid beam repositioning on nanometer scales. A scanning tip/tilt mirror was used to scrutinize larger areas of the sample. The doughnut-shaped excitation PSF was generated by a
Acknowledgments
We thank Steffen J. Sahl and Elisa D’Este (Max Planck Institute for Biophysical Chemistry) for helpful discussions and critical reading of the manuscript.
Footnotes
↵1Y.E. and H.T. contributed equally to this work.
- ↵2To whom correspondence should be addressed. Email: shell{at}mpibpc.mpg.de.
Author contributions: F.B. and S.W.H. designed research; Y.E. and H.T. performed research; H.T. and K.C.G. contributed new reagents/analytic tools; Y.E., H.T., K.C.G., and F.B. analyzed data; and Y.E., F.B., and S.W.H. wrote the paper.
Reviewers: T.H., Johns Hopkins University; and B.S., University of Zurich.
Conflict of interest statement: The Max Planck Society holds patents on selected embodiments and procedures of MINFLUX, benefitting S.W.H., F.B., K.C.G., and Y.E.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1801672115/-/DCSupplemental.
- Copyright © 2018 the Author(s). Published by PNAS.
This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
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