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


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.

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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 and by request.

Code availability

The code used to analyse the data is available at


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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



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.

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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.

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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).

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