Abstract

We present a new method to directly measure and correct the aberrations introduced when imaging through thick biological tissue. A Shack-Hartmann wavefront sensor is used to directly measure the wavefront error induced by a Drosophila embryo. The wavefront measurements are taken by seeding the embryo with fluorescent microspheres used as “artificial guide-stars.” The wavefront error is corrected in ten millisecond steps by applying the inverse to the wavefront error on a micro-electro-mechanical deformable mirror in the image path of the microscope. The results show that this new approach is capable of improving the Strehl ratio by 2 times on average and as high as 10 times when imaging through 100 μm of tissue. The results also show that the isoplanatic half-width is approximately 19 μm resulting in a corrected field of view 38 μm in diameter around the guide-star.

© 2010 OSA

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2010 (3)

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

S. Guldbrand, C. Simonsson, M. Goksör, M. Smedh, and M. B. Ericson, “Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin,” Opt. Express 18(15), 15289–15302 (2010).
[CrossRef] [PubMed]

2007 (2)

M. Schwertner, M. J. Booth, and T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228(1), 97–102 (2007).
[CrossRef] [PubMed]

M. J. Booth, “Adaptive optics in microscopy,” Philos. Trans. R. Soc. London, Ser. A 365(1861), 2829–2843 (2007).
[CrossRef]

2006 (2)

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

C. R. Vogel and Q. Yang, “Modeling, simulation, and open-loop control of a continuous facesheet MEMS deformable mirror,” J. Opt. Soc. Am. A 23(5), 1074–1081 (2006).
[CrossRef]

2005 (1)

A. DeMarais, D. Oldis, and J. M. Quattro, “Matrotrophic Transfer of Fluorescent Microspheres in Poeciliid Fishes,” Copeia 2005(3), 632–636 (2005).
[CrossRef]

2004 (2)

M. Schwertner, M. J. Booth, M. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef]

M. Feierabend, M. Rückel, and W. Denk, “Coherence-gated wave-front sensing in strongly scattering samples,” Opt. Lett. 29(19), 2255–2257 (2004).
[CrossRef] [PubMed]

2003 (2)

2002 (1)

J. L. Beverage, R. V. Shack, and M. R. Descour, “Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor,” J. Microsc. 205(1), 61–75 (2002).
[CrossRef] [PubMed]

1999 (1)

1997 (2)

J. Liang, D. R. Williams, and D. T. Miller, “Supernormal vision and high-resolution retinal imaging through adaptive optics,” J. Opt. Soc. Am. A 14(11), 2884–2892 (1997).
[CrossRef]

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

1996 (1)

A. Dunn and R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2(4), 898–905 (1996).
[CrossRef]

1994 (2)

R. F. Kalpin, D. R. Daily, and W. Sullivan, “Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo,” Biotechniques 17(4), 730–733 (1994).
[PubMed]

J. Liang*, B. Grimm, S. Goelz, and J. F. Bille, “Objective measurement of wave aberrations of the human eye with the use of a Hartmann-Shack wave-front sensor,” J. Opt. Soc. Am. A 11(7), 1949 (1994).
[CrossRef]

1977 (1)

A. Van Helden, “The Invention of the Telescope,” Trans. Am. Phil. Soc. 67, 20–21 (1977).

1953 (1)

H. W. Babcock, “The possibility of compensating astronomical seeing,” Publ. Astron. Soc. Pac. 65, 229–236 (1953).
[CrossRef]

Olivier, Joel

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Agard, David

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Azucena, O.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Azucena, Oscar

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Babcock, H. W.

H. W. Babcock, “The possibility of compensating astronomical seeing,” Publ. Astron. Soc. Pac. 65, 229–236 (1953).
[CrossRef]

Beverage, J. L.

J. L. Beverage, R. V. Shack, and M. R. Descour, “Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor,” J. Microsc. 205(1), 61–75 (2002).
[CrossRef] [PubMed]

Bille, J. F.

Booth, M. J.

M. J. Booth, “Adaptive optics in microscopy,” Philos. Trans. R. Soc. London, Ser. A 365(1861), 2829–2843 (2007).
[CrossRef]

M. Schwertner, M. J. Booth, and T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228(1), 97–102 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef]

Brase, J. M.

Cao, J.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Cao, Jian

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Crest, J.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Crest, Justin

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Daily, D. R.

R. F. Kalpin, D. R. Daily, and W. Sullivan, “Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo,” Biotechniques 17(4), 730–733 (1994).
[PubMed]

Dainty, J. C.

DeMarais, A.

A. DeMarais, D. Oldis, and J. M. Quattro, “Matrotrophic Transfer of Fluorescent Microspheres in Poeciliid Fishes,” Copeia 2005(3), 632–636 (2005).
[CrossRef]

Denk, W.

Descour, M. R.

J. L. Beverage, R. V. Shack, and M. R. Descour, “Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor,” J. Microsc. 205(1), 61–75 (2002).
[CrossRef] [PubMed]

Diaz Santana Haro, L.

Don Gavel, S.

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Don Gavel, S. O.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Dunn, A.

A. Dunn and R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2(4), 898–905 (1996).
[CrossRef]

Ericson, M. B.

Feierabend, M.

Fusco, T.

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Gavel, D. T.

Gerhart, J. C.

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

Goelz, S.

Goksör, M.

Grimm, B.

Guldbrand, S.

Kalpin, R. F.

R. F. Kalpin, D. R. Daily, and W. Sullivan, “Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo,” Biotechniques 17(4), 730–733 (1994).
[PubMed]

Kam, Zvi

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Kner, P.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Kner, Peter

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Kubby,

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Kubby, J.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

Larabell, C. A.

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

Liang, J.

Liang*, J.

Lisa Poyneer, A.

Michau, V.

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Miller, D. T.

Moon, R. T.

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

Neil, M. A.

M. Schwertner, M. J. Booth, M. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef]

Nicolle, M.

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Oldis, D.

A. DeMarais, D. Oldis, and J. M. Quattro, “Matrotrophic Transfer of Fluorescent Microspheres in Poeciliid Fishes,” Copeia 2005(3), 632–636 (2005).
[CrossRef]

Poyneer, L. A.

Quattro, J. M.

A. DeMarais, D. Oldis, and J. M. Quattro, “Matrotrophic Transfer of Fluorescent Microspheres in Poeciliid Fishes,” Copeia 2005(3), 632–636 (2005).
[CrossRef]

Richards-Kortum, R.

A. Dunn and R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2(4), 898–905 (1996).
[CrossRef]

Rousset, G.

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Rowning, B. A.

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

Rückel, M.

Schwertner, M.

M. Schwertner, M. J. Booth, and T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228(1), 97–102 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef]

Sedat, John

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Shack, R. V.

J. L. Beverage, R. V. Shack, and M. R. Descour, “Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor,” J. Microsc. 205(1), 61–75 (2002).
[CrossRef] [PubMed]

Simonsson, C.

Smedh, M.

Sullivan, W.

O. Azucena, J. Cao, J. Crest, W. Sullivan, P. Kner, S. O. Don Gavel, and J. Kubby, “Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction,” Proc. SPIE 7595, 75950I (2010).
[CrossRef]

R. F. Kalpin, D. R. Daily, and W. Sullivan, “Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo,” Biotechniques 17(4), 730–733 (1994).
[PubMed]

Sullivan, William

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

Thomas, S.

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Tokovinin, A.

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Van Helden, A.

A. Van Helden, “The Invention of the Telescope,” Trans. Am. Phil. Soc. 67, 20–21 (1977).

Vogel, C. R.

Wells, J.

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

Williams, D. R.

Wilson, T.

M. Schwertner, M. J. Booth, and T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228(1), 97–102 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef]

Wu, M.

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
[CrossRef] [PubMed]

Yang, Q.

Appl. Opt. (1)

Biotechniques (1)

R. F. Kalpin, D. R. Daily, and W. Sullivan, “Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo,” Biotechniques 17(4), 730–733 (1994).
[PubMed]

Copeia (1)

A. DeMarais, D. Oldis, and J. M. Quattro, “Matrotrophic Transfer of Fluorescent Microspheres in Poeciliid Fishes,” Copeia 2005(3), 632–636 (2005).
[CrossRef]

IEEE J. Sel. Top. Quantum Electron. (1)

A. Dunn and R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2(4), 898–905 (1996).
[CrossRef]

J. Biomed. Opt. (1)

Peter Kner, Jian Cao, Oscar Azucena, Justin Crest, Zvi Kam, John Sedat, David Agard, S. Don Gavel, Joel Olivier, Kubby, and William Sullivan, “Optical Aberrations in Drosophila Embryos,” submitted to J. Biomed. Opt. (2010).

J. Microsc. (3)

M. Schwertner, M. J. Booth, and T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228(1), 97–102 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. Neil, and T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213(1), 11–19 (2004).
[CrossRef]

J. L. Beverage, R. V. Shack, and M. R. Descour, “Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor,” J. Microsc. 205(1), 61–75 (2002).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (4)

Mon. Not. R. Astron. Soc. (1)

S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau, and G. Rousset, “Comparison of centroid computation algorithms in a Shack-Hartmann sensor,” Mon. Not. R. Astron. Soc. 371(1), 323–336 (2006).
[CrossRef]

Opt. Express (1)

Opt. Lett. (2)

Philos. Trans. R. Soc. London, Ser. A (1)

M. J. Booth, “Adaptive optics in microscopy,” Philos. Trans. R. Soc. London, Ser. A 365(1861), 2829–2843 (2007).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (1)

B. A. Rowning, J. Wells, M. Wu, J. C. Gerhart, R. T. Moon, and C. A. Larabell, “Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs,” Proc. Natl. Acad. Sci. U.S.A. 94(4), 1224–1229 (1997).
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Figures (9)

Fig. 1
Fig. 1

Microscope set up with a Deformable Mirror (DM) and a Shack-Hartmann Wavefront Sensor (SHWS). Dichroic filter (D) allows the laser light to be focused onto the sample. Beam Splitter (BS) allows for both the Science Camera (SC) and the SHWS to simultaneously see the fluorescent microsphere.

Fig. 2
Fig. 2

The absorption and emission curves are shown by the dashed and solid lines, respectively. The edge of the source filter is the red line. The emission filter has 90% transmission for wavelengths greater than 641 nm.

Fig. 3
Fig. 3

Combination of a differential interference contrast image (DIC) and a confocal image of injected microspheres in fruit fly embryo 40 microns below the surface of the embryo.

Fig. 4
Fig. 4

A wavefront measurement from a 1 µm fluorescent microsphere embedded 45 µm below the surface of a Drosophila embryo using a 20X (0.40 NA) objective lens with tip, tilt and focus subtracted. The x and y axis are scaled to the sub-aperture diameter. The legend is scaled in microns.

Fig. 5
Fig. 5

Zernike coefficient values for the wavefront shown in Fig. 4. Focus and astigmatism are labeled.

Fig. 6
Fig. 6

PSF analysis. (a) Calculated using a flat wavefront. (b) Calculated using the wavefront shown in Fig. 4. (c) Calculated by removing the first 14 Zernike’s of Fig. 4. (d) Cross-sectional view of a. (e) Cross-sectional view of b. (f) Cross-sectional view of c.

Fig. 7
Fig. 7

Zernike statistical data for the measurements in Table 1. Figures (a) and (b) are the mean of the absolute value for each Zernike mode for the 20X (0.40 NA) and 40X (0.75 NA) objective lenses, respectively. Figures (c) and (d) are the root-mean-square value for each Zernike mode for the 20X (0.40 NA) and 40X (0.75 NA) lenses, respectively.

Fig. 8
Fig. 8

Adaptive optics microscope loop correction steps. Figure 8(a) shows an uncorrected image of the fluorescent microsphere. Figure 8(e) shows the result of closing the loop by using a loop gain factor of 0.4. The length of the bar in Fig. 8(c) is equal to the diffraction limit of the 40X (0.75 NA) objective lens, 0.45 µm. The bead was located 100 um beneath the surface of the embryo.

Fig. 9
Fig. 9

(a) Initial wavefront measurement. (b) Closed-loop DM wavefront. The legend is scaled in percent wavelength at 650 [nm].

Tables (2)

Tables Icon

Table 1 Statistical data for 20X (0.40 NA) and 40X (0.75 NA) objectives. Peak-to-Valley (PV), Root-Mean-Square (RMS), Strelh (S), Strehl after correcting first 14 Zernike’s (S(14))

Tables Icon

Table 2 Isoplanatic angle measurements for the 40X magnification, 0.75 NA objective lens

Equations (4)

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P S F ( x , y ) = | F T { P ( x ' , y ' ) exp ( i 2 π λ w ( x ' , y ' ) ) } | 2 ξ = x λ f , η = y λ f | F T { P ( x ' , y ' ) } | 2 ξ = 0 , η = 0
E ( s ) = W ( s ) D ( s ) = W ( s ) H ( s ) = W ( s ) 1 + K D ( s ) G ( s )
S r e l a t i v e = I p e a k , e I p e a k , a
σ θ 2 = ( φ ( X , 0 ) φ ( X , θ 0 ) ) 2 = 1 rad 2

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