Skip to main content

Thank you for visiting 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.

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


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.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: A simple model of optical focus formation.
Figure 2: Correction of aberrations caused by refractive index mismatch.
Figure 3: Correction of aberrations induced by 250-μm-thick fixed mouse brain slices.
Figure 4: AO correction on a 1-μm diameter bead under a 250-μm fixed brain slice, using different variations of our pupil segmentation algorithm.
Figure 5: Aberration correction at the bottom of an antibody-labeled 300-μm-thick fixed mouse brain slice with beam deflections measured by image correlation.


  1. Ji, N. et al. Advances in the speed and resolution of light microscopy. Curr. Opin. Neurobiol. 18, 605–616 (2008).

    Article  CAS  Google Scholar 

  2. Gibson, S.F. & Lanni, F. Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy. J. Opt. Soc. Am. A 8, 1601–1613 (1991).

    Article  CAS  Google Scholar 

  3. Kam, Z. et al. Computational adaptive optics for live three-dimensional biological imaging. Proc. Natl. Acad. Sci. USA 98, 3790–3795 (2001).

    Article  CAS  Google Scholar 

  4. Schwertner, M. et al. Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry. J. Microsc. 213, 11–19 (2004).

    Article  CAS  Google Scholar 

  5. Schwertner, M., Booth, M.J. & Wilson, T. Characterizing specimen induced aberrations for high NA adaptive optical microscopy. Opt. Express 12, 6540–6552 (2004).

    Article  CAS  Google Scholar 

  6. Babcock, H.W. Adaptive optics revisited. Science 249, 253–257 (1990).

    Article  CAS  Google Scholar 

  7. Tyson, R.K. Principles of adaptive optics (Academic Press, Inc., San Diego, 1991).

  8. Booth, M.J. Adaptive optics in microscopy. Philos. Transact. A Math. Phys. Eng. Sci. 365, 2829–2843 (2007).

    Article  Google Scholar 

  9. Liang, J., Williams, D.R. & Miller, D.T. Supernormal vision and high-resolution retinal imaging through adaptive optics. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 14, 2884–2892 (1997).

    Article  CAS  Google Scholar 

  10. Rueckel, M., Mack-Bucher, J.A. & Denk, W. Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing. Proc. Natl. Acad. Sci. USA 103, 17137–17142 (2006).

    Article  CAS  Google Scholar 

  11. Artal, P. et al. Odd aberrations and double-pass measurements of retinal image quality. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 12, 195–201 (1995).

    Article  CAS  Google Scholar 

  12. Porter, J. et al. eds. Adaptive optics for vision science (John Wiley & Sons, Inc., Hoboken, New Jersey, USA, 2006).

  13. Born, M. & Wolf, E. Principles of Optics (Cambridge University Press, Cambridge, England, 1999).

  14. Goodman, J.W. Introduction to Fourier optics 3rd ed. (Roberts & Company Publishers, Englewood, Colorado, USA, 2005).

  15. Richards, B. & Wolf, E. Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system. Proc. R. Soc. Lond. A Math. Phys. Sci. 253, 358–379 (1959).

    Google Scholar 

  16. Wang, J.Y. & Silva, D.E. Wave-front interpretation with Zernike polynomials. Appl. Opt. 19, 1510–1518 (1980).

    Article  CAS  Google Scholar 

  17. Fernandez, E.J. et al. Adaptive optics with a magnetic deformable mirror: applications in the human eye. Opt. Express 14, 8900–8917 (2006).

    Article  Google Scholar 

  18. Devaney, N. et al. Characterisation of MEMs mirrors for use in atmospheric and ocular wavefront correction. Proc. SPIE 6888, 688802 (2008).

    Article  Google Scholar 

  19. Panagopoulou, S.I. & Neal, D.R. Zonal matrix iterative method for wavefront reconstruction from gradient measurements. J. Refract. Surg. 21, S563–S569 (2005).

    PubMed  Google Scholar 

  20. Southwell, W.H. Wave-front estimation from wave-front slope measurements. J. Opt. Soc. Am. 70, 998–1006 (1980).

    Article  Google Scholar 

  21. Neil, M.A.A. et al. Adaptive aberration correction in a two-photon microscope. J. Microsc. 200, 105–108 (2000).

    Article  Google Scholar 

  22. Booth, M.J. et al. Adaptive aberration correction in a confocal microscope. Proc. Natl. Acad. Sci. USA 99, 5788–5792 (2002).

    Article  CAS  Google Scholar 

  23. Booth, M.J., Neil, M.A.A. & Wilson, T. New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19, 2112–2120 (2002).

    Article  Google Scholar 

  24. Débarre, D. et al. Adaptive optics for structured illumination microscopy. Opt. Express 16, 9290–9305 (2008).

    Article  Google Scholar 

  25. Débarre, D. et al. Image-based adaptive optics for two-photon microscopy. Opt. Lett. 34, 2495–2497 (2009).

    Article  Google Scholar 

  26. Booth, M.J. Wavefront sensorless adaptive optics for large aberrations. Opt. Lett. 32, 5–7 (2007).

    Article  Google Scholar 

  27. Debarre, D., Booth, M.J. & Wilson, T. Image based adaptive optics through optimisation of low spatial frequencies. Opt. Express 15, 8176–8190 (2007).

    Article  Google Scholar 

  28. Hardy, J.W. Adaptive Optics for Astronomical Telescopes (Oxford University Press, Oxford, UK, 1998).

  29. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  Google Scholar 

  30. Oheim, M. et al. Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J. Neurosci. Methods 111, 29–37 (2001).

    Article  CAS  Google Scholar 

  31. Sheppard, C.J.R. & Gu, M. Aberration compensation in confocal microscopy. Appl. Opt. 30, 3563–3568 (1991).

    Article  CAS  Google Scholar 

Download references


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

Authors and Affiliations



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.

Corresponding author

Correspondence to Na Ji.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18 (PDF 8285 kb)

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. (AVI 3930 kb)

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. (AVI 1858 kb)

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. (AVI 4439 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ji, N., Milkie, D. & Betzig, E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat Methods 7, 141–147 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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