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Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues

Nature Methods volume 7, pages 141147 (2010) | Download Citation



Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction–limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 μm.

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We thank our colleagues at Janelia Farm Research Campus, Howard Hughes Medical Institute, B. Shields, A. Hu, W. Amir, R. Kerr, J. Truman, M. Hooks andJ. Makara for help with sample preparation, J. Osborne and S. Bassin for help with machining and T. Sato and T. Planchon for helpful discussions.

Author information


  1. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.

    • Na Ji
    •  & Eric Betzig
  2. Coleman Technologies, Inc., Unit 113, Chadds Ford, Pennsylvania, USA.

    • Daniel E Milkie


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N.J. and E.B. designed the project; N.J., D.E.M. and E.B. developed the instrument control program; N.J. performed the experiments; and N.J. and E.B. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Na Ji.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–18


  1. 1.

    Supplementary Movie 1

    Spatial light modulator patterns (right) and resulting images (left) acquired during implementation of our pupil segmentation based AO algorithm with independent subregion masks and direct phase measurement.

  2. 2.

    Supplementary Movie 2

    Rotating three-dimensional view of integrated intensity projections from a field of 500-nm-diameter fluorescent beads in water before (4× display gain) and after correction for system aberration.

  3. 3.

    Supplementary Movie 3

    Rotating three-dimensional view of integrated intensity projections from a field of 500-nm-diameter fluorescent beads in air before (7× display gain) and after AO correction.

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