Abstract

For many microscopy applications, millimeters-long free working distances (LWD) are required. However, the high resolution and contrast of LWD objectives operated in air are lost when introducing glass and/or liquid with the sample. We propose to use spatial light modulation to correct for such beam aberrations caused by refractive index mismatches. Focusing a monochromatic laser beam with a 10 mm working distance air objective (50×, 0.5 NA) through air, glass, and water, we manage to restore a sharp, intense focus (FWHM<2λ) by adaptive beam phase shaping. Our approach offers a practical and cost-effective route to high resolution and contrast microscopy using LWD air objectives, extending their usage beyond applications in air.

© 2012 Optical Society of America

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References

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  1. H. Gundlach, “Phase contrast and differential interference contrast instrumentation and applications in cell, developmental, and marine biology,” Opt. Eng. 32, 3223–3228(1993).
    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]

2012

T. A. Nenasheva, T. Carter, and G. I. Mashanov, “Automatic tracking of individual migrating cells using low-magnification dark-field microscopy,” J. Microsc. 246, 83–88 (2012).
[CrossRef]

J. M. Bélisle, L. A. Levin, and S. Costantino, “High-content neurite development study using optically patterned substrates,” PLoS One 7, e35911 (2012).
[CrossRef]

J. Scrimgeour and J. E. Curtis, “Aberration correction in wide-field fluorescence microscopy by segmented-pupil image interferometry,” Opt. Express 20, 14534–14541 (2012).
[CrossRef]

T. Wang, A. F. W. van der Steen, and G. van Soest, “Numerical analysis of astigmatism correction in gradient refractive index lens based optical coherence tomography catheters,” Appl. Opt. 51, 5244–5252 (2012).
[CrossRef]

2011

P. P. Mondal and A. Diaspro, “Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy,” Sci. Rep. 1, 149 (2011).
[CrossRef]

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev. 5, 81–101 (2011).
[CrossRef]

B. P. Cumming, A. Jesacher, M. J. Booth, T. Wilson, and M. Gu, “Adaptive aberration compensation for three-dimensional micro-fabrication of photonic crystals in lithium niobate,” Opt. Express 19, 9419–9425 (2011).
[CrossRef]

D. E. Milkie, E. Betzig, and N. Ji, “Pupil-segmentation-based adaptive optical microscopy with full-pupil illumination,” Opt. Lett. 36, 4206–4208 (2011).
[CrossRef]

2010

M. Shaw, S. Hall, S. Knox, R. Stevens, and C. Paterson, “Characterization of deformable mirrors for spherical aberration correction in optical sectioning microscopy,” Opt. Express 18, 6900–6913 (2010).
[CrossRef]

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

P. Kner, J. Sedat, D. Agard, and Z. Kam, “High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing,” J. Microsc. 237, 136–147 (2010).
[CrossRef]

J. J. Schwartz, S. Stavrakis, and S. R. Quake, “Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability,” Nat. Nanotechnol. 5, 127–132 (2010).
[CrossRef]

2009

2008

2007

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

2002

M. Booth, M. Neil, R. Juskaitis, and T. Wilson, “Adaptive aberration correction in a confocal microscope,” Proc. Natl. Acad. Sci. U.S.A. 99, 5788–5792 (2002).
[CrossRef]

2001

R. J. Cook, A. Azzopardi, I. D. Thompson, and T. F. Watson, “Real-time confocal imaging, during active air abrasion—substrate cutting,” J. Microsc. 203, 199–207 (2001).
[CrossRef]

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

1993

H. Gundlach, “Phase contrast and differential interference contrast instrumentation and applications in cell, developmental, and marine biology,” Opt. Eng. 32, 3223–3228(1993).
[CrossRef]

Agard, D.

P. Kner, J. Sedat, D. Agard, and Z. Kam, “High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing,” J. Microsc. 237, 136–147 (2010).
[CrossRef]

Azzopardi, A.

R. J. Cook, A. Azzopardi, I. D. Thompson, and T. F. Watson, “Real-time confocal imaging, during active air abrasion—substrate cutting,” J. Microsc. 203, 199–207 (2001).
[CrossRef]

Bélisle, J. M.

J. M. Bélisle, L. A. Levin, and S. Costantino, “High-content neurite development study using optically patterned substrates,” PLoS One 7, e35911 (2012).
[CrossRef]

Bernet, S.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev. 5, 81–101 (2011).
[CrossRef]

Betzig, E.

Booth, M.

M. Booth, M. Neil, R. Juskaitis, and T. Wilson, “Adaptive aberration correction in a confocal microscope,” Proc. Natl. Acad. Sci. U.S.A. 99, 5788–5792 (2002).
[CrossRef]

Booth, M. J.

Cambi, A.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

can Putten, E. G.

Carter, T.

T. A. Nenasheva, T. Carter, and G. I. Mashanov, “Automatic tracking of individual migrating cells using low-magnification dark-field microscopy,” J. Microsc. 246, 83–88 (2012).
[CrossRef]

Cižmár, T.

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

Cook, R. J.

R. J. Cook, A. Azzopardi, I. D. Thompson, and T. F. Watson, “Real-time confocal imaging, during active air abrasion—substrate cutting,” J. Microsc. 203, 199–207 (2001).
[CrossRef]

Costantino, S.

J. M. Bélisle, L. A. Levin, and S. Costantino, “High-content neurite development study using optically patterned substrates,” PLoS One 7, e35911 (2012).
[CrossRef]

Cumming, B. P.

Curtis, J. E.

de Bakker, B.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

de Lange, F.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

Dholakia, K.

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

Diaspro, A.

P. P. Mondal and A. Diaspro, “Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy,” Sci. Rep. 1, 149 (2011).
[CrossRef]

Figdor, C. G.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

Garcia-Parajo, M.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

Girkin, J. M.

Gu, M.

Gundlach, H.

H. Gundlach, “Phase contrast and differential interference contrast instrumentation and applications in cell, developmental, and marine biology,” Opt. Eng. 32, 3223–3228(1993).
[CrossRef]

Hall, S.

Huijbens, R.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

Inoue, T.

Itoh, H.

Jesacher, A.

B. P. Cumming, A. Jesacher, M. J. Booth, T. Wilson, and M. Gu, “Adaptive aberration compensation for three-dimensional micro-fabrication of photonic crystals in lithium niobate,” Opt. Express 19, 9419–9425 (2011).
[CrossRef]

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev. 5, 81–101 (2011).
[CrossRef]

Ji, N.

Juskaitis, R.

M. Booth, M. Neil, R. Juskaitis, and T. Wilson, “Adaptive aberration correction in a confocal microscope,” Proc. Natl. Acad. Sci. U.S.A. 99, 5788–5792 (2002).
[CrossRef]

Kam, Z.

P. Kner, J. Sedat, D. Agard, and Z. Kam, “High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing,” J. Microsc. 237, 136–147 (2010).
[CrossRef]

Kner, P.

P. Kner, J. Sedat, D. Agard, and Z. Kam, “High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing,” J. Microsc. 237, 136–147 (2010).
[CrossRef]

Knox, S.

Levin, L. A.

J. M. Bélisle, L. A. Levin, and S. Costantino, “High-content neurite development study using optically patterned substrates,” PLoS One 7, e35911 (2012).
[CrossRef]

Mashanov, G. I.

T. A. Nenasheva, T. Carter, and G. I. Mashanov, “Automatic tracking of individual migrating cells using low-magnification dark-field microscopy,” J. Microsc. 246, 83–88 (2012).
[CrossRef]

Matsumoto, N.

Maurer, C.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev. 5, 81–101 (2011).
[CrossRef]

Mazilu, M.

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

Milkie, D. E.

Mondal, P. P.

P. P. Mondal and A. Diaspro, “Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy,” Sci. Rep. 1, 149 (2011).
[CrossRef]

Mosk, A. P.

E. G. can Putten, I. M. Vellekoop, and A. P. Mosk, “Spatial amplitude and phase modulation using commercial twisted nematic LCDs,” Appl. Opt. 47, 2076–2081 (2008).
[CrossRef]

I. M. Vellekoop and A. P. Mosk, “Phase control algorithms for focusing light through turbid media,” Opt. Commun. 281, 3071–3080 (2008).
[CrossRef]

Neil, M.

M. Booth, M. Neil, R. Juskaitis, and T. Wilson, “Adaptive aberration correction in a confocal microscope,” Proc. Natl. Acad. Sci. U.S.A. 99, 5788–5792 (2002).
[CrossRef]

Nenasheva, T. A.

T. A. Nenasheva, T. Carter, and G. I. Mashanov, “Automatic tracking of individual migrating cells using low-magnification dark-field microscopy,” J. Microsc. 246, 83–88 (2012).
[CrossRef]

Paterson, C.

Poland, S. P.

Quake, S. R.

J. J. Schwartz, S. Stavrakis, and S. R. Quake, “Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability,” Nat. Nanotechnol. 5, 127–132 (2010).
[CrossRef]

Rensen, W.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

Ritsch-Marte, M.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev. 5, 81–101 (2011).
[CrossRef]

Schwartz, J. J.

J. J. Schwartz, S. Stavrakis, and S. R. Quake, “Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability,” Nat. Nanotechnol. 5, 127–132 (2010).
[CrossRef]

Scrimgeour, J.

Sedat, J.

P. Kner, J. Sedat, D. Agard, and Z. Kam, “High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing,” J. Microsc. 237, 136–147 (2010).
[CrossRef]

Shaw, M.

Stavrakis, S.

J. J. Schwartz, S. Stavrakis, and S. R. Quake, “Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability,” Nat. Nanotechnol. 5, 127–132 (2010).
[CrossRef]

Stevens, R.

Thompson, I. D.

R. J. Cook, A. Azzopardi, I. D. Thompson, and T. F. Watson, “Real-time confocal imaging, during active air abrasion—substrate cutting,” J. Microsc. 203, 199–207 (2001).
[CrossRef]

van der Steen, A. F. W.

van Hulst, N.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

van Soest, G.

Vellekoop, I. M.

E. G. can Putten, I. M. Vellekoop, and A. P. Mosk, “Spatial amplitude and phase modulation using commercial twisted nematic LCDs,” Appl. Opt. 47, 2076–2081 (2008).
[CrossRef]

I. M. Vellekoop and A. P. Mosk, “Phase control algorithms for focusing light through turbid media,” Opt. Commun. 281, 3071–3080 (2008).
[CrossRef]

Wang, T.

Watson, T. F.

R. J. Cook, A. Azzopardi, I. D. Thompson, and T. F. Watson, “Real-time confocal imaging, during active air abrasion—substrate cutting,” J. Microsc. 203, 199–207 (2001).
[CrossRef]

Wilson, T.

B. P. Cumming, A. Jesacher, M. J. Booth, T. Wilson, and M. Gu, “Adaptive aberration compensation for three-dimensional micro-fabrication of photonic crystals in lithium niobate,” Opt. Express 19, 9419–9425 (2011).
[CrossRef]

M. Booth, M. Neil, R. Juskaitis, and T. Wilson, “Adaptive aberration correction in a confocal microscope,” Proc. Natl. Acad. Sci. U.S.A. 99, 5788–5792 (2002).
[CrossRef]

Wright, A. J.

Appl. Opt.

J. Cell Sci.

F. de Lange, A. Cambi, R. Huijbens, B. de Bakker, W. Rensen, M. Garcia-Parajo, N. van Hulst, and C. G. Figdor, “Cell biology beyond the diffraction limit: near-field scanning optical microscopy,” J. Cell Sci. 114, 4154–4160 (2001).

J. Microsc.

T. A. Nenasheva, T. Carter, and G. I. Mashanov, “Automatic tracking of individual migrating cells using low-magnification dark-field microscopy,” J. Microsc. 246, 83–88 (2012).
[CrossRef]

R. J. Cook, A. Azzopardi, I. D. Thompson, and T. F. Watson, “Real-time confocal imaging, during active air abrasion—substrate cutting,” J. Microsc. 203, 199–207 (2001).
[CrossRef]

P. Kner, J. Sedat, D. Agard, and Z. Kam, “High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing,” J. Microsc. 237, 136–147 (2010).
[CrossRef]

Laser Photon. Rev.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photon. Rev. 5, 81–101 (2011).
[CrossRef]

Nat. Nanotechnol.

J. J. Schwartz, S. Stavrakis, and S. R. Quake, “Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability,” Nat. Nanotechnol. 5, 127–132 (2010).
[CrossRef]

Nat. Photonics

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

Opt. Commun.

I. M. Vellekoop and A. P. Mosk, “Phase control algorithms for focusing light through turbid media,” Opt. Commun. 281, 3071–3080 (2008).
[CrossRef]

Opt. Eng.

H. Gundlach, “Phase contrast and differential interference contrast instrumentation and applications in cell, developmental, and marine biology,” Opt. Eng. 32, 3223–3228(1993).
[CrossRef]

Opt. Express

Opt. Lett.

Phil. Trans. R. Soc. A

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

PLoS One

J. M. Bélisle, L. A. Levin, and S. Costantino, “High-content neurite development study using optically patterned substrates,” PLoS One 7, e35911 (2012).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A.

M. Booth, M. Neil, R. Juskaitis, and T. Wilson, “Adaptive aberration correction in a confocal microscope,” Proc. Natl. Acad. Sci. U.S.A. 99, 5788–5792 (2002).
[CrossRef]

Sci. Rep.

P. P. Mondal and A. Diaspro, “Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy,” Sci. Rep. 1, 149 (2011).
[CrossRef]

Other

R. L. Price and W. G. Jerome, eds., Basic Confocal Microscopy (Springer, 2011).

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Figures (8)

Fig. 1.
Fig. 1.

Focusing with an air immersion 10-mm-long WD objective, O1, (NA=0.5, 50×) through media of different refractive indices, ni, causes focus aberrations of the order of 1.8 mm in the nominal focal plane (NFP, small dash). The planes for aberration correction with spatial light modulation P1 and P2 (thick dash) are located 1.5 mm below the NFP at the smallest beam waist. See text for details.

Fig. 2.
Fig. 2.

Schematics of experimental setup. He–Ne, 632.8 nm cw laser; SLM, amplitude and phase spatial light modulator; BS, beam splitter; O1, air-immersion objective for focusing and for reflection experiments, WD=10mm, NA=0.5, 50×; O2, oil-immersion objective for transmission experiments, WD=0.13mm, NA=1.4, 100×; G, glass window; W, water; S, sample (150 μm thick glass slide in transmission mode, aluminium mirror in reflection mode); CCD, charge-coupled device camera.

Fig. 3.
Fig. 3.

Gaussian beam plane wave, transmission mode. Left: before phase correction; middle: after phase correction; right: correction phase pattern. (a) Air, (b) glass window, (c) glass window/water at P1, and (d) glass window/water at P2.

Fig. 4.
Fig. 4.

P1, transmission mode. Left: before phase correction; middle: after phase correction; right: correction phase pattern. (a) Center rays, (b) middle rays, and (c) outer rays as selected with amplitude mask (insets in left column; white: pixel on, black: pixel off).

Fig. 5.
Fig. 5.

P2, transmission mode. Left: before phase correction; middle: after phase correction; right: correction phase pattern. (a) Center rays and (b) middle rays as selected with amplitude mask (insets in left column; white: pixel on, black: pixel off).

Fig. 6.
Fig. 6.

Gaussian beam plane wave, reflection mode. Left: before phase correction; middle: after phase correction; right: correction phase pattern. (a) Air, (b) glass window, (c) glass window/water at P1, and (d) glass window/water at P2.

Fig. 7.
Fig. 7.

P1, reflection mode. Left: before phase correction; middle: after phase correction; right: correction phase pattern. (a) Center rays, (b) middle rays, and (c) outer rays as selected with amplitude mask (insets in left column: white, pixel on; black, pixel off).

Fig. 8.
Fig. 8.

P2, reflection mode. Left: before phase correction; middle: after phase correction; right: correction phase pattern. (a) Center rays and (b) middle rays as selected with amplitude mask (insets in left column: white, pixel on; black, pixel off).

Tables (2)

Tables Icon

Table 1. Focus Size (FWHM) after Aberration Correction in Nanometers

Tables Icon

Table 2. Intensity Ratios, I, after/before Focus Correctiona

Metrics