Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Six-dimensional single-molecule imaging with isotropic resolution using a multi-view reflector microscope

Abstract

Imaging of both the positions and orientations of single fluorophores, termed single-molecule orientation-localization microscopy, is a powerful tool for the study of biochemical processes. However, the limited photon budget associated with single-molecule fluorescence makes high-dimensional imaging with isotropic, nanoscale spatial resolution a formidable challenge. Here we realize a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the three-dimensional (3D) positions and 3D orientations of single molecules, with precisions of 10.9 nm and 2.0° over a 1.5-μm depth range. The raMVR microscope achieves 6D super-resolution imaging of Nile red molecules transiently bound to lipid-coated spheres, accurately resolving their spherical morphology, despite refractive-index mismatch. By observing the rotational dynamics of Nile red, raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labelling. Finally, we demonstrate 6D imaging of cell membranes, where the orientations of specific fluorophores reveal heterogeneity in membrane fluidity. With its nearly isotropic 3D spatial resolution and orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with exceptional detail.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Concept of the raMVR SMOLM.
Fig. 2: Best possible orientation and localization precision of raMVR compared to other methods.
Fig. 3: 6D nanoscopic imaging of spherical SLBs.
Fig. 4: 6D nanoscopic imaging of the amyloid–lipid interaction.
Fig. 5: 6D nanoscopic imaging of MC540 molecules bound to the membranes of an HEK-293T cell.

Data availability

The data underlying this study are openly available from OSF at https://osf.io/s8m4x/?view_only=f65ebe6038cc4bd79a8438be84e35309 and by request.

Code availability

The code used to analyse the data is available at https://osf.io/s8m4x/?view_only=f65ebe6038cc4bd79a8438be84e35309.

References

  1. Norregaard, K., Metzler, R., Ritter, C. M., Berg-Sørensen, K. & Oddershede, L. B. Manipulation and motion of organelles and single molecules in living cells. Chem. Rev. 117, 4342–4375 (2017).

    Article  Google Scholar 

  2. Stone, M. B., Shelby, S. A. & Veatch, S. L. Super-resolution microscopy: shedding light on the cellular plasma membrane. Chem. Rev. 117, 7457–7477 (2017).

    Article  Google Scholar 

  3. Lyon, A. S., Peeples, W. B. & Rosen, M. K. A framework for understanding the functions of biomolecular condensates across scales. Nat. Rev. Mol. Cell Biol. 22, 215–235 (2021).

    Article  Google Scholar 

  4. Emenecker, R. J., Holehouse, A. S. & Strader, L. C. Biological phase separation and biomolecular condensates in plants. Annu. Rev. Plant Biol. 72, 17–46 (2021).

    Article  Google Scholar 

  5. Moerner, W. E. & Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535–2538 (1989).

    Article  Google Scholar 

  6. Moerner, W. E. Viewpoint: single molecules at 31: what’s next? Nano Lett. 20, 8427–8429 (2020).

    Article  Google Scholar 

  7. Asenjo, A. B., Krohn, N. & Sosa, H. Configuration of the two kinesin motor domains during ATP hydrolysis. Nat. Struct. Mol. Biol. 10, 836–842 (2003).

    Article  Google Scholar 

  8. Beausang, J. F., Shroder, D. Y., Nelson, P. C. & Goldman, Y. E. Tilting and wobble of myosin V by high-speed single-molecule polarized fluorescence microscopy. Biophys. J. 104, 1263–1273 (2013).

    Article  Google Scholar 

  9. Backer, A. S., Lee, M. Y. & Moerner, W. E. Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements. Optica 3, 659–666 (2016).

    Article  Google Scholar 

  10. Backer, A. S. et al. Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA. Sci. Adv. 5, eaav1083 (2019).

    Article  Google Scholar 

  11. Hulleman, C. N. et al. Simultaneous orientation and 3D localization microscopy with a vortex point spread function. Nat. Commun. 12, 5934 (2021).

    Article  Google Scholar 

  12. Curcio, V., Alemán-Castañeda, L. A., Brown, T. G., Brasselet, S. & Alonso, M. A. Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation. Nat. Commun. 11, 5307 (2020).

    Article  Google Scholar 

  13. Rimoli, C. V., Valades-Cruz, C. A., Curcio, V., Mavrakis, M. & Brasselet, S. 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. Nat. Commun. 13, 301 (2022).

    Article  Google Scholar 

  14. Ding, T., Wu, T., Mazidi, H., Zhang, O. & Lew, M. D. Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils. Optica 7, 602–607 (2020).

    Article  Google Scholar 

  15. Ding, T. & Lew, M. D. Single-molecule localization microscopy of 3D orientation and anisotropic wobble using a polarized vortex point spread function. J. Phys. Chem. B 125, 12718–12729 (2021).

    Article  Google Scholar 

  16. Lu, J., Mazidi, H., Ding, T., Zhang, O. & Lew, M. D. Single molecule 3D orientation imaging reveals nanoscale compositional heterogeneity in lipid membranes. Angew. Chem. Int. Ed. 59, 17572–17579 (2020).

    Article  Google Scholar 

  17. Zhang, O., Zhou, W., Lu, J., Wu, T. & Lew, M. D. Resolving the three-dimensional rotational and translational dynamics of single molecules using radially and azimuthally polarized fluorescence. Nano Lett. 22, 1024–1031 (2022).

    Article  Google Scholar 

  18. Backlund, M. P., Lew, M. D., Backer, A. S., Sahl, S. J. & Moerner, W. E. The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging. ChemPhysChem 15, 587–599 (2014).

    Article  Google Scholar 

  19. Zhang, O. & Lew, M. D. Quantum limits for precisely estimating the orientation and wobble of dipole emitters. Phys. Rev. Res. 2, 033114 (2020).

    Article  Google Scholar 

  20. Zhang, O. & Lew, M. D. Single-molecule orientation localization microscopy II: a performance comparison. J. Opt. Soc. Am. A 38, 288–297 (2021).

    Article  Google Scholar 

  21. Beckwith, J. S. & Yang, H. Information bounds in determining the 3D orientation of a single emitter or scatterer using point-detector-based division-of-amplitude polarimetry. J. Chem. Phys. 155, 144110 (2021).

    Article  Google Scholar 

  22. Wu, T., Lu, J. & Lew, M. D. Dipole-spread-function engineering for simultaneously measuring the 3D orientations and 3D positions of fluorescent molecules. Optica 9, 505–511 (2022).

    Article  Google Scholar 

  23. Mlodzianoski, M. J. et al. Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections. Nat. Methods 15, 583–586 (2018).

    Article  Google Scholar 

  24. Xu, F. et al. Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval. Nat. Methods 17, 531–540 (2020).

    Article  Google Scholar 

  25. Backer, A. S., Backlund, M. P., Lew, M. D. & Moerner, W. E. Single-molecule orientation measurements with a quadrated pupil. Opt. Lett. 38, 1521–1523 (2013).

    Article  Google Scholar 

  26. Backer, A. S., Backlund, M. P., von Diezmann, A. R., Sahl, S. J. & Moerner, W. E. A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy. Appl. Phys. Lett. 104, 193701 (2014).

    Article  Google Scholar 

  27. Zhang, O., Lu, J., Ding, T. & Lew, M. D. Imaging the three-dimensional orientation and rotational mobility of fluorescent emitters using the Tri-spot point spread function. Appl. Phys. Lett. 113, 031103 (2018).

    Article  Google Scholar 

  28. Zhang, O. & Lew, M. D. Single-molecule orientation localization microscopy I: fundamental limits. J. Opt. Soc. Am. A 38, 277–287 (2021).

    Article  Google Scholar 

  29. Novotny, L. & Hecht, B. Principles of Nano-Optics (Cambridge Univ. Press, 2012).

  30. Backlund, M. P. et al. Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask. Nat. Photon. 10, 459–462 (2016).

    Article  Google Scholar 

  31. Thorsen, R. Ø., Hulleman, C. N., Rieger, B. & Stallinga, S. Photon efficient orientation estimation using polarization modulation in single-molecule localization microscopy. Biomed. Opt. Express 13, 2835–2858 (2022).

    Article  Google Scholar 

  32. Böhmer, M. & Enderlein, J. Orientation imaging of single molecules by wide-field epifluorescence microscopy. J. Opt. Soc. Am. B 20, 554–559 (2003).

    Article  Google Scholar 

  33. Lieb, M. A., Zavislan, J. M. & Novotny, L. Single-molecule orientations determined by direct emission pattern imaging. J. Opt. Soc. Am. B 21, 1210–1215 (2004).

    Article  Google Scholar 

  34. Axelrod, D. Fluorescence excitation and imaging of single molecules near dielectric-coated and bare surfaces: a theoretical study. J. Microsc. 247, 147–160 (2012).

    Article  Google Scholar 

  35. Lew, M. D. & Moerner, W. E. Azimuthal polarization filtering for accurate, precise and robust single-molecule localization microscopy. Nano Lett. 14, 6407–6413 (2014).

    Article  Google Scholar 

  36. Moon, T. K. & Stirling, W. C. Mathematical Methods and Algorithms for Signal Processing (Prentice Hall, 2000).

  37. Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

    Article  Google Scholar 

  38. Haass, C. & Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid β-peptide. Nat. Rev. Mol. Cell Biol. 8, 101–112 (2007).

  39. Iadanza, M. G., Jackson, M. P., Hewitt, E. W., Ranson, N. A. & Radford, S. E. A new era for understanding amyloid structures and disease. Nat. Rev. Mol. Cell Biol. 19, 755–773 (2018).

    Article  Google Scholar 

  40. Butterfield, S. M. & Lashuel, H. A. Amyloidogenic protein-membrane interactions: mechanistic insight from model systems. Angew. Chem. Int. Ed. 49, 5628–5654 (2010).

    Article  Google Scholar 

  41. Bode, D. C., Freeley, M., Nield, J., Palma, M. & Viles, J. H. Amyloid-β oligomers have a profound detergent-like effect on lipid membrane bilayers, imaged by atomic force and electron microscopy. J. Biol. Chem. 294, 7566–7572 (2019).

    Article  Google Scholar 

  42. Hampel, H. et al. Core candidate neurochemical and imaging biomarkers of Alzheimer’s disease. Alzheimers Dement. 4, 38–48 (2008).

  43. Choucair, A., Chakrapani, M., Chakravarthy, B., Katsaras, J. & Johnston, L. Preferential accumulation of Aβ(1-42) on gel phase domains of lipid bilayers: an AFM and fluorescence study. Biochim. Biophys. Acta 1768, 146–154 (2007).

    Article  Google Scholar 

  44. Hane, F., Drolle, E., Gaikwad, R., Faught, E. & Leonenko, Z. Amyloid-β aggregation on model lipid membranes: an atomic force microscopy study. J. Alzheimers Dis. 26, 485–494 (2011).

  45. Kuo, C. & Hochstrasser, R. M. Super-resolution microscopy of lipid bilayer phases. J. Am. Chem. Soc. 133, 4664–4667 (2011).

    Article  Google Scholar 

  46. Moon, S. et al. Spectrally resolved, functional super-resolution microscopy reveals nanoscale compositional heterogeneity in live-cell membranes. J. Am. Chem. Soc. 139, 10944–10947 (2017).

    Article  Google Scholar 

  47. Klymchenko, A. S. Solvatochromic and fluorogenic dyes as environment-sensitive probes: design and biological applications. Acc. Chem. Res. 50, 366–375 (2017).

    Article  Google Scholar 

  48. Lee, S.-C. et al. Fluorescent molecular rotors for viscosity sensors. Chem. A Eur. J. 24, 13706–13718 (2018).

    Article  Google Scholar 

  49. Danylchuk, D. I., Moon, S., Xu, K. & Klymchenko, A. S. Switchable solvatochromic probes for live cell super resolution imaging of plasma membrane organization. Angew. Chem. Int. Ed. 58, 14920–14924 (2019).

    Article  Google Scholar 

  50. Harauz, G. & van Heel, M. Exact filters for general geometry three dimensional reconstruction. Optik 73, 146–156 (1986).

    Google Scholar 

  51. Banterle, N., Bui, K. H., Lemke, E. A. & Beck, M. Fourier ring correlation as a resolution criterion for super-resolution microscopy. J. Struct. Biol. 183, 363–367 (2013).

    Article  Google Scholar 

  52. Dragsten, P. R. & Webb, W. W. Mechanism of the membrane potential sensitivity of the fluorescent membrane probe merocyanine 540. Biochemistry 17, 5228–5240 (1978).

    Article  Google Scholar 

  53. Verkman, A. S. & Frosch, M. P. Temperature-jump studies of merocyanine 540 relaxation kinetics in lipid bilayer membranes. Biochemistry 24, 7117–7122 (1985).

    Article  Google Scholar 

  54. Yu, H. & Hui, S.-W. Merocyanine 540 as a probe to monitor the molecular packing of phosphatidylcholine: a monolayer epifluorescence microscopy and spectroscopy study. Biochim. Biophys. Acta 1107, 245–254 (1992).

    Article  Google Scholar 

  55. Wilson-Ashworth, H. A. et al. Differential detection of phospholipid fluidity, order and spacing by fluorescence spectroscopy of bis-pyrene, prodan, nystatin and merocyanine 540. Biophys. J. 91, 4091–4101 (2006).

    Article  Google Scholar 

  56. Lagerberg, J. W., Kallen, K.-J., Haest, C. W., VanSteveninck, J. & Dubbelman, T. M. Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells. Biochim. Biophys. Acta 1235, 428–436 (1995).

    Article  Google Scholar 

  57. Verkman, A. S. Mechanism and kinetics of merocyanine 540 binding to phospholipid membranes. Biochemistry 26, 4050–4056 (1987).

    Article  Google Scholar 

  58. Sims, R. R. et al. Single molecule light field microscopy. Optica 7, 1065–1072 (2020).

    Article  Google Scholar 

  59. Gustavsson, A.-K., Petrov, P. N. & Moerner, W. E. Light sheet approaches for improved precision in 3D localization-based super-resolution imaging in mammalian cells [Invited]. Opt. Express 26, 13122–13147 (2018).

    Article  Google Scholar 

  60. Ponjavic, A., Ye, Y., Laue, E., Lee, S. F. & Klenerman, D. Sensitive light-sheet microscopy in multiwell plates using an AFM cantilever. Biomed. Opt. Express 9, 5863–5880 (2018).

    Article  Google Scholar 

  61. Zelger, P. et al. Three-dimensional localization microscopy using deep learning. Opt. Express 26, 33166–33179 (2018).

    Article  Google Scholar 

  62. Möckl, L., Roy, A. R. & Moerner, W. E. Deep learning in single-molecule microscopy: fundamentals, caveats and recent developments [Invited]. Biomed. Opt. Express 11, 1633–1661 (2020).

    Article  Google Scholar 

  63. Nehme, E. et al. DeepSTORM3D: dense 3D localization microscopy and PSF design by deep learning. Nat. Methods 17, 734–740 (2020).

    Article  Google Scholar 

  64. Nehme, E. et al. Learning optimal wavefront shaping for multi-channel imaging. IEEE Trans. Pattern Anal. Mach. Intell. 43, 2179–2192 (2021).

    Article  Google Scholar 

  65. Wu, T., Lu, P., Rahman, M. A., Li, X. & Lew, M. D. Deep-SMOLM: deep learning resolves the 3D orientations and 2D positions of overlapping single molecules with optimal nanoscale resolution. Opt. Express 30, 36761–36773 (2022).

    Article  Google Scholar 

  66. Speiser, A. et al. Deep learning enables fast and dense single-molecule localization with high accuracy. Nat. Methods 18, 1082–1090 (2021).

    Article  Google Scholar 

  67. Bayerl, T. & Bloom, M. Physical properties of single phospholipid bilayers adsorbed to micro glass beads. A new vesicular model system studied by 2H-nuclear magnetic resonance. Biophys. J. 58, 357–362 (1990).

    Article  Google Scholar 

Download references

Acknowledgements

We thank A. Backer and V. Acosta for helpful discussions in designing the raMVR microscope. Aβ42 peptide was synthesized and purified by J. I. Elliott (ERI Amyloid Laboratory, Oxford, CT). Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases under grant no. R21AI163985 to M.D.V., by the National Science Foundation under grant no. ECCS-1653777 to M.D.L., and by the National Institute of General Medical Sciences of the National Institutes of Health under grant no. R35GM124858 to M.D.L.

Author information

Authors and Affiliations

Authors

Contributions

O.Z. and M.D.L. designed and built the raMVR imaging system. Z.G., Y.H. and M.D.V. prepared fixed HEK-293T cells. T.W. developed the protocol for preparing lipid-coated silica spheres. O.Z. performed experiments and analysed the data.

Corresponding author

Correspondence to Matthew D. Lew.

Ethics declarations

Competing interests

A patent application covering the raMVR imaging technology reported in this manuscript has been filed by Washington University (O.Z. and M.D.L. as inventors, application no. PCT/US2021/063071). The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Photonics thanks Sophie Brasselet, Bernd Rieger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Pyramid mirror designs.

(a) Alignment of the pyramidal mirrors and (b-e) dimensions of the (b) air pyramid, (c) one sector of the air pyramid, (d) mount for glass pyramid, and (e) glass pyramid. (i) Isometric view, (ii) top view, and (iii) side view of the mirrors and mount. Length unit: mm.

Extended Data Fig. 2 Simulation of rays propagating in the MVR system.

A total of 100 rays, randomly oriented within the imaging system’s numerical aperture and originating from random positions within the intermediate image plane (IIP) are shown. (a) Three-dimensional view of rays propagating within one of the polarized imaging channels. (b-d) Position of each ray at the (b) IIP, (c) glass pyramid, (d) air pyramid, and (e) detector. Scale bars: 50 mm in (a), 1 mm in (b,c), 5 mm in (d,e).

Extended Data Fig. 3 Light propagation within the second 4f system of the raMVR microscope.

(a) When folding mirrors FM1-4 are out of the emission path, detectors C1 and C2 observe the (i) radially and (ii) azimuthally polarized image plane. Colourbar: photons; scale bar: 2 μm. (b) When FM1 and FM3 are in the emission path and mirror M3 is properly aligned, detector C2 captures the (i) radially polarized BFP. Similarly, when FM2 and FM4 are in the emission path, detector C1 captures the (ii) azimuthally polarized BFP. Grid size: 1 cm; colourbar: normalized intensity; scale bar: 1 mm.

Extended Data Fig. 4 Image size comparison of CHIDO, the vortex DSF, raPol standard DSF and raMVR.

(a-d) Images and (e) line profiles of (a) CHIDO (x -polarized channel), (b) the Vortex DSF, (c) raPol standard DSF (radially polarized channel), and (d) raMVR (azimuthally polarized channel) for isotropic emitters located at z = 250 nm, z = 750 nm (best focus), and z = 1250 nm. The nominal focal plane zf = 1200 nm. Colourbar: normalized intensity; scale bar: 500 nm. (e) Line profiles of the DSFs in (a-d). Yellow: CHIDO; green: Vortex DSF; gray: raPol standard DSF; red: raMVR.

Extended Data Fig. 5 6D nanoscopic imaging of MC540 molecules bound to the membranes of another HEK-293T cell.

(a,b) Super-resolved images of two representative cells. Colours represent the estimated (a) axial position z and (b) azimuthal angle ϕ. Inset: summed diffraction-limited images from all imaging channels captured under lower excitation power. Scale bar: 2 μm. (c) yz - and (d,e) xy -views of boxed regions in (b). Lines are oriented and colour-coded according to the measured (c) polar angle θ, (d) wobble angle Ω, and (e) azimuthal angle ϕ. Their lengths are proportional to (c) \(\sqrt{{\mu }_{y}^{2}+{\mu }_{z}^{2}}\)) and (d,e) \(\sin \theta\). Scale arrows: 2 μm in (a,b), 500 nm in (c-e). (d) Insets show the distribution of measured wobble angle Ω within the yellow and white boxed areas (i,ii) and a side view of yellow and white boxed areas (i). Three-dimensional animations of the localizations in (c-e) are shown in Movie S5. (f) MC540 tilt Δϕ relative to the membrane surface within the white boxed areas in (d,e).

Extended Data Fig. 6 6D nanoscopic imaging of NR4A molecules bound to the membranes of HEK-293T cells.

(a-d) Super-resolved images of two representative cells. Colours represent the estimated (a,c) axial position z and (b,d) azimuthal angle ϕ. Insets: summed diffraction-limited images from all imaging channels captured under lower excitation power. Scale bar: 2 μm. (e,f,h) The zoomed regions of interest in (b,d). Lines are oriented and colour-coded according to the measured azimuthal angle ϕ. Their lengths are proportional to \(\sin \theta\). Scale bar: 500 nm. (g,j) Distributions of measured azimuthal angle from the membrane Δϕ within the white boxed areas in (e,f,h).

Supplementary information

Supplementary Information

Supplementary Sections 1–5, Figs. 1–32 and Tables 1–5.

Supplementary Video 1

Fluorescent beads on the surface of a glass coverslip (z = 0), embedded in lens immersion oil, axially scanned from zf = −500 nm to zf = 500 nm. Colour scale, photons; scale, 2 μm.

Supplementary Video 2

Three-dimensional view and cross-sectional images of Nile red localizations on the 1,000-nm-radius lipid-coated sphere in Fig. 3. Localizations are depicted as points at left and as line segments matching the measured molecule orientations at right. Colour scale, polar angle θ in degrees (left) and azimuthal angle ϕ in degrees (right).

Supplementary Video 3

Three-dimensional view of Nile red localizations on 350-nm-radius lipid-coated spheres incubated without (day 0) and with amyloid beta (Aβ42; Fig. 4). Colour scale, azimuthal angle ϕ in degrees.

Supplementary Video 4

Two-dimensional (xy) view of merocyanine 540 localizations along the HEK-293T cell membrane in Extended Data Fig. 5a, and a sliding window showing yz cross-sections. Localizations are depicted as points at left and as line segments matching the measured molecule orientations at right. Colour scale, axial position z in nm.

Supplementary Video 5

Three-dimensional view of merocyanine 540 localizations along the HEK-293T cell membrane in Fig. 5c–e and Extended Data Fig. 5c–e. Localizations are depicted as line segments matching the measured molecule orientations. Colour scale, polar angle ϕ in degrees (left), wobble solid angle Ω in sr (middle) and azimuthal angle ϕ in degrees (right).

Supplementary Video 6

Blinking merocyanine 540 molecules along the HEK-293T cell in Fig. 5a. Dashed lines represent where the cell is in contact with coverslip (bottom-right area in each channel). The nominal focal plane is placed at zf = 1,200 nm. Colour scale, photons; scale, 5 μm.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, O., Guo, Z., He, Y. et al. Six-dimensional single-molecule imaging with isotropic resolution using a multi-view reflector microscope. Nat. Photon. (2022). https://doi.org/10.1038/s41566-022-01116-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41566-022-01116-6

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing