Abstract

We present a new technique for the correction of optical aberrations in wide-field fluorescence microscopy. Segmented-Pupil Image Interferometry (SPII) uses a liquid crystal spatial light modulator placed in the microscope’s pupil plane to split the wavefront originating from a fluorescent object into an array of individual beams. Distortion of the wavefront arising from either system or sample aberrations results in displacement of the images formed from the individual pupil segments. Analysis of image registration allows for the local tilt in the wavefront at each segment to be corrected with respect to a central reference. A second correction step optimizes the image intensity by adjusting the relative phase of each pupil segment through image interferometry. This ensures that constructive interference between all segments is achieved at the image plane. Improvements in image quality are observed when Segmented-Pupil Image Interferometry is applied to correct aberrations arising from the microscope’s optical path.

© 2012 OSA

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2011

W. Supatto, T. V. Truong, D. Débarre, and E. Beaurepaire, “Advances in multiphoton microscopy for imaging embryos,” Curr. Opin. Genet. Dev. 21(5), 538–548 (2011).
[CrossRef] [PubMed]

G. Hall, G. C. Spalding, P. J. Campagnola, J. G. White, and K. W. Eliceiri, “Fast localized wavefront correction using area-mapped phase-shift interferometry,” Opt. Lett. 36(15), 2892–2894 (2011).
[CrossRef] [PubMed]

2010

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

T. Cizmar, M. Mazilu, and K. Dholakia, “In situ wavefront correction and its application to micromanipulation,” Nat. Photonics 4(6), 388–394 (2010).
[CrossRef]

R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack-Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010).
[CrossRef]

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

2009

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

2008

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

2007

I. M. Vellekoop and A. P. Mosk, “Focusing coherent light through opaque strongly scattering media,” Opt. Lett. 32(16), 2309–2311 (2007).
[CrossRef] [PubMed]

M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. 365(1861), 2829–2843 (2007).
[CrossRef] [PubMed]

2005

2004

2003

2002

1994

1990

1988

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Arnaud, J.

Artal, P.

Azam, F.

Beaurepaire, E.

W. Supatto, T. V. Truong, D. Débarre, and E. Beaurepaire, “Advances in multiphoton microscopy for imaging embryos,” Curr. Opin. Genet. Dev. 21(5), 538–548 (2011).
[CrossRef] [PubMed]

Betzig, E.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Bille, J. F.

Booth, M. J.

M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. 365(1861), 2829–2843 (2007).
[CrossRef] [PubMed]

Bowman, R. W.

R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack-Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010).
[CrossRef]

Campagnola, P. J.

Carroll, J.

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

Cizmar, T.

T. Cizmar, M. Mazilu, and K. Dholakia, “In situ wavefront correction and its application to micromanipulation,” Nat. Photonics 4(6), 388–394 (2010).
[CrossRef]

Coarer, E. L.

Débarre, D.

W. Supatto, T. V. Truong, D. Débarre, and E. Beaurepaire, “Advances in multiphoton microscopy for imaging embryos,” Curr. Opin. Genet. Dev. 21(5), 538–548 (2011).
[CrossRef] [PubMed]

Dholakia, K.

T. Cizmar, M. Mazilu, and K. Dholakia, “In situ wavefront correction and its application to micromanipulation,” Nat. Photonics 4(6), 388–394 (2010).
[CrossRef]

Dubis, A. M.

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

Duncan, J. L.

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

Eliceiri, K. W.

Fernandez, E.

Fernández, E.

Fuchs, E.

Girkin, J. M.

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

Godara, P.

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

Goelz, S.

Grimm, B.

Hall, G.

Jaffe, J.

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Keller, P. J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Lelievre, G.

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Lelièvre, G.

Liang, J.

Llebaria, A.

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Long, R.

Manzanera, S.

Mazilu, M.

T. Cizmar, M. Mazilu, and K. Dholakia, “In situ wavefront correction and its application to micromanipulation,” Nat. Photonics 4(6), 388–394 (2010).
[CrossRef]

Milkie, D. E.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Mosk, A. P.

Nieto, J. L.

Nieto, J.-L.

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Oron, D.

Padgett, M. J.

R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack-Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010).
[CrossRef]

Poland, S.

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

Prieto, P.

Roorda, A.

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

Salmon, D.

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Sebag, J.

Silberberg, Y.

Spalding, G. C.

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Supatto, W.

W. Supatto, T. V. Truong, D. Débarre, and E. Beaurepaire, “Advances in multiphoton microscopy for imaging embryos,” Curr. Opin. Genet. Dev. 21(5), 538–548 (2011).
[CrossRef] [PubMed]

Tal, E.

Thouvenot, E.

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Truong, T. V.

W. Supatto, T. V. Truong, D. Débarre, and E. Beaurepaire, “Advances in multiphoton microscopy for imaging embryos,” Curr. Opin. Genet. Dev. 21(5), 538–548 (2011).
[CrossRef] [PubMed]

Vellekoop, I. M.

White, J. G.

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Wright, A. J.

R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack-Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010).
[CrossRef]

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

Astron. Astrophys.

G. Lelievre, J.-L. Nieto, E. Thouvenot, D. Salmon, and A. Llebaria, “Very high resolution imaging using sub-pupil apertures, recentering and selection of short exposures,” Astron. Astrophys. 200, 301–311 (1988).

Curr. Opin. Biotechnol.

J. M. Girkin, S. Poland, and A. J. Wright, “Adaptive optics for deeper imaging of biological samples,” Curr. Opin. Biotechnol. 20(1), 106–110 (2009).
[CrossRef] [PubMed]

Curr. Opin. Genet. Dev.

W. Supatto, T. V. Truong, D. Débarre, and E. Beaurepaire, “Advances in multiphoton microscopy for imaging embryos,” Curr. Opin. Genet. Dev. 21(5), 538–548 (2011).
[CrossRef] [PubMed]

J. Opt.

R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack-Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010).
[CrossRef]

J. Opt. Soc. Am. A

Nat. Methods

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[CrossRef] [PubMed]

Nat. Photonics

T. Cizmar, M. Mazilu, and K. Dholakia, “In situ wavefront correction and its application to micromanipulation,” Nat. Photonics 4(6), 388–394 (2010).
[CrossRef]

Opt. Express

Opt. Lett.

Optom. Vis. Sci.

P. Godara, A. M. Dubis, A. Roorda, J. L. Duncan, and J. Carroll, “Adaptive optics retinal imaging: emerging clinical applications,” Optom. Vis. Sci. 87(12), 930–941 (2010).
[CrossRef] [PubMed]

Philos. Transact. A Math. Phys. Eng. Sci.

M. J. Booth, “Adaptive optics in microscopy,” Philos. Transact. A Math. Phys. Eng. Sci. 365(1861), 2829–2843 (2007).
[CrossRef] [PubMed]

Science

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Other

J. W. Hardy, Adaptive Optics for Astronomical Telescopes (Oxford University Press, New York, 1988).

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Figures (5)

Fig. 1
Fig. 1

Illustrates the effect of a distorted wavefront on the registration of images captured from different parts of the pupil plane (left). An appropriate grating function applied to the probe segment corrects the observed registration error (right).

Fig. 2
Fig. 2

Evolution of interference fringes in the image of a 0.5 μm fluorescent microsphere (a) as the phase difference (ΔΦ) between the reference segment and a neighboring probe segment is shifted between 0 and 2π radians. (b) Intensity profiles passing horizontally through the center of the fluorescent microsphere’s image.

Fig. 3
Fig. 3

Comparison of methods for optimizing the path difference between a probe segment and the reference. (a) Direct tracking of the intensity at the centroid of a microsphere, (b) Tracking the average value in the top 10% of pixel intensities, and (c) tracking the peak value of the cross correlation between the captured images and the original reference image. In each case the data is displayed with the fitted curve used to extract the optimal phase adjustment for the corrected pupil segment.

Fig. 4
Fig. 4

Correction of system aberrations. (a) Shows the kinoform produced by the SPII correction procedure. (b) Shows the result of imaging of 0.5 μm fluorescent microspheres using no aberration correction, SPII correction, and the SLM’s factory correction respectively. For each condition an image of a cluster of microspheres is shown alongside a through-focus image series. Scale bar 1 μm. (c) Shows a comparison between the intensity profiles of a single 0.5 μm fluorescent microsphere captured using no correction (blue line), the SPII correction (green line) and the SLM factory correction (red line). There are 105 nm per pixel.

Fig. 5
Fig. 5

Illustrates the improvements in visualization of the cytoskeleton in BPAE cells resulting from the application of the SPII correction. (a) no applied correction, (b) SPII correction. Arrows indicate features of interest. Scale bar 5 μm.

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