Abstract

We propose high-quality generation of uniform multiple fluorescence spots (MFS) with a spatial light modulator (SLM) and demonstrate uniform laser scanning in multifocal multiphoton microscopy (MMM). The MFS excitation method iteratively updates a computer-generated hologram (CGH) using correction coefficients to improve the fluorescence intensity distribution in a dye solution whose consistency is uniform. This simple correction method can be applied for calibration of the MMM before observation of living tissue. We experimentally demonstrate an improvement of the uniformity of a 10 × 10 grid of MFS by using a dye solution. After the calibration, we performed laser scanning with two-photon excitation to observe fluorescent polystyrene beads, as well as the gastric gland of a guinea pig specimen.

© 2014 Optical Society of America

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    [CrossRef] [PubMed]
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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] [PubMed]

2013

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

2012

2008

2007

2006

2005

Y. Hayasaki, T Sugimoto, A. Takita, N. Nishida, “Variable holographic femtosecond laser processing by use of a spatial light modulator,” Appl. Phys. Lett. 87, 031101 (2005).
[CrossRef]

2004

O. Ripoll, V. Kettunen, H. P. Herzig, “Review of iterative Fourier-transform algorithms for beam shaping applications,” Opt. Eng. 43, 2549–2556 (2004).
[CrossRef]

2003

2000

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, “High efficiency beam splitter for multifocal mutiphoton microscopy,” Journal of Microscopy 201, 368–376 (2000).
[CrossRef]

1997

N. Yoshikawa, M. Itoh, T. Yatagai, “Adaptive computer-generated hologram using interpolation method,” Opt. Rev. 4, A161–A163 (1997).
[CrossRef]

1994

1992

1990

U. Mahlab, J. Rosen, J. Shamir, “Iterative generation of holograms on spatial light modulators,” Opt. Lett. 15, 556–558 (1990).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Ameer-Beg, S.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Andersen, P.

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, “High efficiency beam splitter for multifocal mutiphoton microscopy,” Journal of Microscopy 201, 368–376 (2000).
[CrossRef]

Ando, T.

Antolini, R.

Bahlmann, K.

Bellve, K.

Bewersdorf, J.

J. Bewersdorf, A. Egner, S. W. Hell, “Multifocal Multi-photon Microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed., 3rd ed. (Springer, 2006), 550–560.
[CrossRef]

Buehler, C.

Choudhury, A.

Coelho, S.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Dandliker, R.

Denk, W.

P. Theer, W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23, 3139–3149 (2006).
[CrossRef]

W. Denk, J. H. Strickler, W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Egner, A.

J. Bewersdorf, A. Egner, S. W. Hell, “Multifocal Multi-photon Microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed., 3rd ed. (Springer, 2006), 550–560.
[CrossRef]

Emiliani, V.

Fantini, S.

Fricke, M.

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, “High efficiency beam splitter for multifocal mutiphoton microscopy,” Journal of Microscopy 201, 368–376 (2000).
[CrossRef]

Froner, E.

Fukuchi, N.

N. Matsumoto, T. Ando, T. Inoue, Y. Ohtake, N. Fukuchi, T. Hara, “Generation of high-quality higher-order Laguerre-Gaussian beams using liquid-crystal-on-silicon spatial light modulators,” J. Opt. Soc. Am. A 25, 1642–1651 (2008).
[CrossRef]

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

Gale, M. T.

Gao, B. Z.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Guillon, M.

Hara, T.

Hasegawa, S.

Hayasaki, Y.

H. Takahashi, S. Hasegawa, Y. Hayasaki, “Holographic femtosecond laser processing using optimal-rotation-angle method with compensation of spatial frequency response of liquid crystal modulator,” Appl. Opt. 46, 5917–5923 (2007).
[CrossRef] [PubMed]

Y. Hayasaki, T Sugimoto, A. Takita, N. Nishida, “Variable holographic femtosecond laser processing by use of a spatial light modulator,” Appl. Phys. Lett. 87, 031101 (2005).
[CrossRef]

Heffer, E. L.

Hell, S. W.

J. Bewersdorf, A. Egner, S. W. Hell, “Multifocal Multi-photon Microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed., 3rd ed. (Springer, 2006), 550–560.
[CrossRef]

Hellweg, D.

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, “High efficiency beam splitter for multifocal mutiphoton microscopy,” Journal of Microscopy 201, 368–376 (2000).
[CrossRef]

Henderson, R.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Herzig, H. P.

O. Ripoll, V. Kettunen, H. P. Herzig, “Review of iterative Fourier-transform algorithms for beam shaping applications,” Opt. Eng. 43, 2549–2556 (2004).
[CrossRef]

D. Prongue, H. P. Herzig, R. Dandliker, M. T. Gale, “Optimized kinoform structures for highly efficient fan-out elements,” Appl. Opt. 31, 5706–5711 (1992).
[CrossRef] [PubMed]

Ianni, F.

Igasaki, Y.

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

Inoue, T.

Itoh, M.

N. Yoshikawa, M. Itoh, T. Yatagai, “Adaptive computer-generated hologram using interpolation method,” Opt. Rev. 4, A161–A163 (1997).
[CrossRef]

Kettunen, V.

O. Ripoll, V. Kettunen, H. P. Herzig, “Review of iterative Fourier-transform algorithms for beam shaping applications,” Opt. Eng. 43, 2549–2556 (2004).
[CrossRef]

Kim, K. H.

Kirber, M.

Kobayashi, Y.

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

N. Mukohzaka, N. Yoshida, H. Toyoda, Y. Kobayashi, T. Hara, “Diffraction efficiency analysis of a parallel-aligned nematic-liquid-crystal spatial light modulator,” Appl. Opt. 33, 2804–2811 (1994).
[CrossRef] [PubMed]

Kosicki, B.

Krstajic, N.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Lee, W. A.

Leonardo, R. D.

Li, D.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Lin, H.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Mahlab, U.

Matsumoto, N.

McGonagle, W.

Moneypenny, J.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Mukohzaka, N.

Nedivi, E.

Ng, T.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Nielsen, T.

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, “High efficiency beam splitter for multifocal mutiphoton microscopy,” Journal of Microscopy 201, 368–376 (2000).
[CrossRef]

Nishida, N.

Y. Hayasaki, T Sugimoto, A. Takita, N. Nishida, “Variable holographic femtosecond laser processing by use of a spatial light modulator,” Appl. Phys. Lett. 87, 031101 (2005).
[CrossRef]

Niu, H.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Ohtake, Y.

Pavone, F. S.

Peng, X.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Poland, S.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Prongue, D.

Qin, W.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Qu, J.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Ragan, T.

Reich, R.

Ripoll, O.

O. Ripoll, V. Kettunen, H. P. Herzig, “Review of iterative Fourier-transform algorithms for beam shaping applications,” Opt. Eng. 43, 2549–2556 (2004).
[CrossRef]

Ronzitti, E.

Rosen, J.

Ruocco, G.

Sacconi, L.

Sars, V. D.

Shamir, J.

Shao, Y.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Shiv, L.

So, P. T. C.

Stein, J.

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Sugimoto, T

Y. Hayasaki, T Sugimoto, A. Takita, N. Nishida, “Variable holographic femtosecond laser processing by use of a spatial light modulator,” Appl. Phys. Lett. 87, 031101 (2005).
[CrossRef]

Taghizadeh, M. R.

Takahashi, H.

Takiguchi, Y.

Takita, A.

Y. Hayasaki, T Sugimoto, A. Takita, N. Nishida, “Variable holographic femtosecond laser processing by use of a spatial light modulator,” Appl. Phys. Lett. 87, 031101 (2005).
[CrossRef]

Takumi, M.

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

Tanaka, H.

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

Theer, P.

Toyoda, H.

Tyndall, D.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Walker, R.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Yatagai, T.

N. Yoshikawa, M. Itoh, T. Yatagai, “Adaptive computer-generated hologram using interpolation method,” Opt. Rev. 4, A161–A163 (1997).
[CrossRef]

Yoshida, N.

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

N. Mukohzaka, N. Yoshida, H. Toyoda, Y. Kobayashi, T. Hara, “Diffraction efficiency analysis of a parallel-aligned nematic-liquid-crystal spatial light modulator,” Appl. Opt. 33, 2804–2811 (1994).
[CrossRef] [PubMed]

Yoshikawa, N.

N. Yoshikawa, M. Itoh, T. Yatagai, “Adaptive computer-generated hologram using interpolation method,” Opt. Rev. 4, A161–A163 (1997).
[CrossRef]

Appl. Opt.

Appl. Phys. B

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, B. Z. Gao, “Multifocal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[CrossRef]

Appl. Phys. Lett.

Y. Hayasaki, T Sugimoto, A. Takita, N. Nishida, “Variable holographic femtosecond laser processing by use of a spatial light modulator,” Appl. Phys. Lett. 87, 031101 (2005).
[CrossRef]

J. Opt. Soc. Am. A

Journal of Microscopy

T. Nielsen, M. Fricke, D. Hellweg, P. Andersen, “High efficiency beam splitter for multifocal mutiphoton microscopy,” Journal of Microscopy 201, 368–376 (2000).
[CrossRef]

Opt. Eng.

O. Ripoll, V. Kettunen, H. P. Herzig, “Review of iterative Fourier-transform algorithms for beam shaping applications,” Opt. Eng. 43, 2549–2556 (2004).
[CrossRef]

Opt. Express

Opt. Lett.

Opt. Rev.

N. Yoshikawa, M. Itoh, T. Yatagai, “Adaptive computer-generated hologram using interpolation method,” Opt. Rev. 4, A161–A163 (1997).
[CrossRef]

Proc. SPIE

T. Inoue, H. Tanaka, N. Fukuchi, M. Takumi, N. Matsumoto, T. Hara, N. Yoshida, Y. Igasaki, Y. Kobayashi, “LCOS spatial light modulator controlled by 12-bit signals for optical phase-only modulation,” Proc. SPIE 6487, 64870Y (2007).
[CrossRef]

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, S. Ameer-Beg, “Multifocal multiphoton microsopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[CrossRef]

Science

W. Denk, J. H. Strickler, W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Other

J. Bewersdorf, A. Egner, S. W. Hell, “Multifocal Multi-photon Microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed., 3rd ed. (Springer, 2006), 550–560.
[CrossRef]

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Figures (8)

Fig. 1
Fig. 1

Block diagram of the proposed method: (a) outer iteration and (b) inner iteration. Parameters in the dashed boxes are stored in memory.

Fig. 2
Fig. 2

Schematic of the experimental MMM setup. The solid lines (red) and the dashed lines (green) represent the excitation beam and the fluorescence light, respectively.

Fig. 3
Fig. 3

Observed 10 × 10 grid of MFS obtained by applying (a) the proposed method and (b) the conventional OC method. (c) Relation between the number of adaptive feedback iterations and the uniformity.

Fig. 4
Fig. 4

(a) The scanner performed a raster scan with the 10 × 10 grid of MFS. The fluorescence spots in the blue area were acquired at 23 × 23 positions. The results of superimposing all these images obtained (b) with the proposed method and(c) with the conventional OC method. The fluorescence intensity distribution of each ROI obtained (d) with the proposed method and (e) with the conventional OC method. These figures indicate that the uniformity is high when the whole area is white.

Fig. 5
Fig. 5

The composite images of 10-μm- and 3-μm-diameter fluorescent beads (a) with the proposed method and (b) with the conventional OC method, respectively. The false-color image obtained from the pseudo-confocal processing (c) with the proposed method and (d) with the conventional OC method, respectively. The insets in Fig. 5(c) and (d) show the magnified view of a single bead. In the inset of Fig. 5(d), we can observe block noise segments caused by non-uniform MFS in the bead. Fluorescence intensity profiles on the same line of Fig. 5(a) and (c) were plotted in Fig. 5(e).

Fig. 6
Fig. 6

Observations of the gastric gland of a guinea pig specimen by using a dual-CCD camera. Composite images obtained (a) with the proposed method and (b) with the conventional OC method. The images after pseudo-confocal processing obtained (c) with the proposed method and (d) with the conventional OC method.

Fig. 7
Fig. 7

Uniformity of fluorescence intensity distribution as function of (a) the number of MFS, L × L, (b) the spot separation d, and (c) the spatial frequency of the CGH. The η value was strongly sensitive to the spatial frequency of the CGH. Figure 7(d) shows the number of outer iteration steps until η became less than 0.05.

Fig. 8
Fig. 8

Observed honeycomb-shaped grid of fluorescent spots obtained by applying the proposed method.

Equations (5)

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v ( k ) ( m ) = v ( k 1 ) ( m ) q ( k 1 ) ( m ) q ( k ) ( m ) 2 n ,
T l ( k ) ( m ) = v ( k ) ( m ) w l ( k ) ( m ) T goal ( m ) ,
w l ( k ) ( m ) = w l 1 ( k ) ( m ) J l 1 ( k ) J l ( k ) ,
σ = 1 q ave m = 1 M [ q ( m ) q ave ] 2 M ,
η = q max q min 2 q ave ,

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