Distance measurements are commonly performed by phase detection based on a lock-in strategy. Super-resolution fluorescence microscopy is still striving to perform axial localization but through entirely different strategies. Here we show that an illumination modulation approach can achieve nanometric axial localization precision without compromising the acquisition time, emitter density or lateral localization precision. The excitation pattern is obtained by shifting tilted interference fringes. The molecular localizations are performed by measuring the relative phase between each fluorophore response and the reference modulated excitation pattern. We designed a fast demodulation scheme compatible with the short emission duration of single emitters. This modulated localization microscopy offers a typical axial localization precision of 6.8 nm over the entire field of view and the axial capture range. Furthermore, the interfering pattern being robust to optical aberrations, a nearly uniform axial localization precision enables imaging of biological samples by up to several micrometres in depth.
The data that supports the images and plots, within this paper and other findings are available from the corresponding author upon reasonable request.
Processing code are based on already published solutions as described in the Supplementary Information.
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P.J. acknowledges Master’s funding from GDR ImaBio and PhD funding from IDEX Paris Saclay (grant no. ANR-11-IDEX-0003-02). M.B. was funded by the Labex PALM (ANR-10-LABX-0039-PALM). We acknowledge the advices of the Centre de Photonique pour la Biologie et les Matériaux to cell culture and labelling. We also thank G. Dupuis for discussion and S. Sreenivas for a careful reading of the manuscript. We thank Abbelight for the free use of NEO software and dSTORM buffers. This work was supported by the AXA research fund, the ANR (grant nos. LABEX WIFI, ANR-10-LABX-24), ANR MSM-modulated super-resolution microscopy (grant no. ANR-17-CE09-0040), the valorization programme of the IDEX Paris Saclay and of Labex PALM.
The CNRS has deposited a patent FR3054321-A1 on the 25 July 2016 to protect this work, currently under international extension. S.L.F, E.F. and N.B. are co-inventors.
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Jouchet, P., Cabriel, C., Bourg, N. et al. Nanometric axial localization of single fluorescent molecules with modulated excitation. Nat. Photonics (2021). https://doi.org/10.1038/s41566-020-00749-9