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Molecular-scale axial localization by repetitive optical selective exposure

Nature Methods volume 18, pages 369–373 (2021)Cite this article

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Abstract

We introduce an axial localization with repetitive optical selective exposure (ROSE-Z) method for super-resolution imaging. By using an asymmetric optical scheme to generate interference fringes, a <2 nm axial localization precision was achieved with only ~3,000 photons, which is an approximately sixfold improvement compared to previous astigmatism methods. Nanoscale three-dimensional and two-color imaging was demonstrated, illustrating how this method achieves superior performance and facilitates the investigation of cellular nanostructures.

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Fig. 1: Working principle and validation of ROSE-Z.
Fig. 2: 3D ROSE-Z imaging of cell samples.
Fig. 3: Multicolor imaging of cell samples with ROSE-Z.

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Data availability

The source data for Figs. 2a and 3b,g,h are provided as supplementary source data. The other data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The MATLAB program for analysis and the modified Gpufit source code are freely available for academic use and are provided as Supplementary software. LabVIEW program for device control and Python program for drift correction during imaging, which are hardware-dependent, are available from the corresponding author upon request.

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Acknowledgements

We thank K. Xiao for help in verifying the beads sample. This work was supported by the National Key Research and Development Program of China (grant no. 2017YFA0505300 to W.J.), the National Natural Science Foundation of China (grant no. 32027901 to T.X., grant no. 91954202 to W.J., grant no. 31730054, 31661143041 and 31127901 to T.X., grant no. 31700743 and 91954201 to L.G.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB37000000 to W.J.), Beijing Municipal Science & Technology Commission no. Z191100007219004 (to T.X.), the instrument development project of CAS (grant no. GJJSTD20180001 to T.X., grant no. YJKYYQ20180069 to W.J.), the Youth Innovation Promotion Association of CAS (grant no. 2013066 to W.J., grant no. 2017135 to L.G.) and Beijing Nova Program (Z202003) from Beijing Municipal Science & Technology Commission.

Author information

Author notes

  1. These authors contributed equally: Lusheng Gu, Yuanyuan Li, Shuwen Zhang.

Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Lusheng Gu, Yuanyuan Li, Shuwen Zhang, Maoge Zhou, Yanhong Xue, Weixing Li, Tao Xu & Wei Ji

  2. Physical Science Laboratory, Huairou National Comprehensive Science Center, Beijing, China

    Lusheng Gu, Yuanyuan Li, Shuwen Zhang, Maoge Zhou, Yanhong Xue, Weixing Li, Tao Xu & Wei Ji

  3. Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, Guangdong, China

    Lusheng Gu, Weixing Li, Tao Xu & Wei Ji

  4. College of Life Science, University of Chinese Academy of Sciences, Beijing, China

    Lusheng Gu, Shuwen Zhang, Maoge Zhou, Yanhong Xue, Tao Xu & Wei Ji

  5. Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Lusheng Gu, Yuanyuan Li, Weixing Li & Wei Ji

  6. School of Future Technology, University of Chinese Academy of Sciences, Beijing, China

    Shuwen Zhang & Tao Xu

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  1. Lusheng Gu
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Contributions

W.J., T.X. and L.G. designed the experiments. W.J. and L.G. developed the ROSE-Z system. L.G. performed the data analysis. Y.L., S.Z. and M.Z. conducted sample preparation and imaging. T.X., W.J., L.G., Y.X. and W.L. interpreted the results. W.J., T.X. and L.G. wrote the manuscript, which was modified by all other authors.

Corresponding authors

Correspondence to Tao Xu or Wei Ji.

Ethics declarations

Competing interests

A CN patent (ZL 201920675223.6) has been issued describing the ROSE detection system used in this work; L.G., W.J. and T.X. are the co-inventors. A patent describing the ROSE-Z system and image analysis in this work has been filed; L.G., W.J. and T.X. are the co-inventors. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Methods thanks Jesse Aaron and the other, anonymous reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 One frame of a raw image from EMCCD in ROSE-Z.

Each frame of raw image with 512*512 pixels contains 4 subimages were indicated by subimage 1–4. Subimages 1–3 were used for the extraction of axial localization in ROSE-Z; subimage 4 was used for the demixing detection in multi-color imaging. The size of each subimage is approximately 220*220 pixels, with a pixel size of 150 nm, yielding a FOV of approximately 33*33 μm2. Scale bar: 10 μm. 5 experiments were repeated independently with similar results.

Extended Data Fig. 2 Detailed optical setup of ROSE-Z.

Laser1: MSL-FN-639, CNI; Laser2: OBIS 405 nm LX, Coherent; AOTF: AOTFnC-400.650, AA; LED: M530L4, Thorlabs; Fiber Coupler: HPUC-23AF-400/700-P-4.5AC-15, OZ; Fiber Collimator: HPUCO-23-400/700-P-6AC, OZ; PM fiber: QPMJ-3AF3S-400-3/125-3AS-3-1, OZ; Half waveplate: WPH05M-633, Thorlabs; EOM: Model 350-80, ConOptics; Beam Expander: GCO-2501, CDH; PBS: PBS251, Thorlabs; DM1, DM2 and DM4: FF552-Di02-25x36, Semrock; DM3: ZT405/488/561/640rpc-v2, Chroma; DM5: ZT405/488/561/647rpc, Chroma; Filter1: FF01-661/20-25, Semrock; Filter2: ZET405/488/561/647m, Chroma; Objective: Uapo N 100X 1.49 N.A. Oil immersion objective, Olympus; CCD1: iXon DU897 EMCCD, Andor; CCD2: Guppy F-033B, Allied Vision Technologies; Resonant Mirror: CRS 4 KHZ, CTI; DAQ: PCI-6733, NI; CPLD: epm570t100c5n, Intel.

Extended Data Fig. 3 Axial localization calibration.

a, The images of 40 nm fluorescent nanosphere under different axial locations (upper) and the orthogonal slices of the PSF (lower). The position scanning was performed by a piezo stage, the step sizes were 25 nm for the upper fluorescent nanosphere images and 5 nm for the lower orthogonal PSF slices, respectively. b, The relationship between PSF width in the XY direction and the axial location. c, The width difference shows a monotonical relationship with the axial location, and a 3-order polynomial fitting curve was used to calculate the rough axial position. d, The intensity of 3 subimages under different axial locations. The calibration result of the system showed 263.3 nm interference fringe period. Scale bar: 2 μm in a. For data in a to d, 5 experiments were repeated independently with similar results.

Extended Data Fig. 4 Photos of the optical setup and the sample.

a, Close up of the chamber ready for STORM imaging, the top of the chamber was covered with another coverslip to create an optical window for illumination and was sealed with Vaseline. b, Close up of the microscope sample stage, showing the sample and the optics in the upper illumination path. The distance between the two coverslips was not restricted as in iPALM and 4Pi-SMS. c, The optical setup was built based on an Olympus IX71 inverted microscope.

Extended Data Fig. 5 Modulation depth analysis across the whole FOV.

a, The modulation depth of the fluorescent nanospheres within the FOV. b, The histogram of the modulation depth in a, indicating high contrast of the interference fringes. Scale bar: 10 μm in a. For data in a and b, 5 experiments were repeated independently with similar results.

Extended Data Fig. 6 Phase and phase difference across the FOV.

a, The scan positions of a fluorescent nanosphere which was used to calculate the phase and phase difference across the FOV. The scan was performed by a piezo stage within a region of 25 * 25 μm2. b, Distribution of three phases of the fluorescent nanosphere at each scanning positions within the FOV, with standard deviation of 7.2, 4.8 and 5.9 degrees, respectively. c, Distribution of the phase difference between phase1 and phase2 showing mean value of 119.9 degrees. d, Distribution of the phase difference between phase1 and phase3 showing mean value of -118.0 degrees (wrapped to a range of -180 to 180 degrees).

Extended Data Fig. 7 Three-dimensional localization distribution of single molecules.

a, Three-dimensional localization distribution of single molecules with ROSE-Z. The results were based on the microtubule network data in Fig. 2. Molecules appeared for more than 10 frames were selected for analysis, and localizations from 474 clusters were aligned by their center-of-mass to generate the overall 3D presentation of the localization distribution. b-d, Localization distribution of single molecules in the X- (b), Y- (c) and Z- (d) directions with ROSE-Z, respectively. The histograms were fitted to a Gaussian function, yielding standard deviation of 8.4 nm in X, 8.7 nm in Y and 3.4 nm in Z, respectively. e, Three-dimensional localization distribution of single molecules with astigmatic method. Molecules appeared for more than 10 frames were selected for analysis, and localizations from 387 clusters were aligned by their center-of-mass to generate the overall 3D presentation of the localization distribution. f-h, Localization distribution of single molecules in the X- (f), Y- (g) and Z- (h) directions with astigmatism, respectively. The histograms were fit to a Gaussian function, yielding standard deviation of 8.8 nm in X, 8.7 nm in Y and 17.6 nm in Z, respectively.

Extended Data Fig. 8 Orthogonal slices of the Fourier Plane Correlation (FPC) of a ROSE-Z image of microtubule network.

a-c, Cross-sections of the qxqy-plane (a), the qxqz-plane (b), and the qyqz-plane (c) of the Fourier Plane Correlation, with resolution threshold contours indicating FPC = 1/7 (black lines). The correlation result indicated resolution of 23.0 nm, 29.8 nm and 20.4 nm in the X- Y- and Z- directions, respectively. Since the FPC show summed effect of localization precision, labeling error, feature of biological structure and system drift, so the resolution in axial direction did not show much advantage to lateral resolution in the results of FPC, and the resolution in X and Y also show greater difference due to the spreading direction of the microtubule network. d, Representation of the 3D microtubule network, with the axial coordinate in false color. Scale bar: 1 μm in d.

Extended Data Fig. 9 Transmission profiles for the conventional and demixing detection.

a, Transmission profiles of the dichroic mirror and emission filters for the conventional detection path. The magenta area shows the combined transmission profile of the dichroic mirror and emission filters. b, Transmission profiles of the dichroic mirror and emission filters for the demixing detection path. The green area shows the combined transmission profile of the dichroic mirror and emission filters. c-d, The emission spectra and the fraction of conventional and demixing detection for each dye used in this work. Spectra were obtained from the respective manufacturers’ websites.

Extended Data Fig. 10 Demonstration of thick cellular ROSE-Z imaging.

a, Color-coded ROSE-Z imaging of Alexa-647 labeled TOM-20 in COS-7 cells. The thickness of the sample was approximately 2.4 μm. b-c, Cross-sections of the corresponding area indicated by the dash line in a. Scale bars: 5 μm in a and 3 μm in b and c. For data in a to c, 3 experiments were repeated independently with similar results.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Notes 1–4.

Reporting Summary

Supplementary Video 1

Two-color ROSE-Z image of microtubule and mitochondria in COS-7 cell.

Supplementary Video 2

ROSE-Z image of thick mitochondria structures in COS-7 cell.

Supplementary Software 1

MATLAB code for reconstruction with demo data.

Supplementary Software 2

Modified Gpufit source code for Gaussian fitting.

Source data

Source Data Fig. 2

Raw reconstruction data in Fig. 2a.

Source Data Fig. 3

Raw data in Fig. 3b,g,h.

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Gu, L., Li, Y., Zhang, S. et al. Molecular-scale axial localization by repetitive optical selective exposure. Nat Methods 18, 369–373 (2021). https://doi.org/10.1038/s41592-021-01099-2

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