Abstract

Abstract

We introduce a multiphoton microscope for high-speed three-dimensional (3D) fluorescence imaging. The system combines parallel illumination by a multifocal multiphoton microscope (MMM) with parallel detection via a segmented high-sensitivity charge-couple device (CCD) camera. The instrument consists of a Ti-sapphire laser illuminating a microlens array that projects 36 foci onto the focal plane. The foci are scanned using a resonance scanner and imaged with a custom-made CCD camera. The MMM increases the imaging speed by parallelizing the illumination; the CCD camera can operate at a frame rate of 1428 Hz while maintaining a low read noise of 11 electrons per pixel by dividing its chip into 16 independent segments for parallelized readout. We image fluorescent specimens at a frame rate of 640 Hz. The calcium wave of fluo3 labeled cardiac myocytes is measured by imaging the spontaneous contraction of the cells in a 0.625 second sequence movie, consisting of 400 single images.

© 2007 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).
  2. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655 (1998).
    [CrossRef]
  3. A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
    [CrossRef]
  4. J. Bewersdorf, A. Egner, and S. W. Hell, "Multifocal Multi-Photon Microscopy" in Handbook of Biological Confocal Microscopy, J. Pawley, ed., (Springer, 2006), pp. 550-560.
  5. M. Petran, M. Hadravsky, M. D. Egger, and R. Galambos, "Tandem scanning reflected-light microscope," J. Opt. Soc. Am. 58, 661-664 (1968).
    [CrossRef]
  6. G. S. Kino and T. R. Corle, "Confocal scanning optical microscopy," Phys. Today 42, 55-62 (1989).
    [CrossRef]
  7. I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).
  8. M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
    [CrossRef] [PubMed]
  9. T. P. Padera, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte endothelial interactions," Mol. Imaging,"  1, 9-15 (2002).
    [CrossRef]
  10. K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
    [CrossRef]
  11. T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
    [CrossRef] [PubMed]
  12. T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
    [CrossRef] [PubMed]
  13. B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
  14. A. Egner and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A 17, 1192 (2000).
    [CrossRef]
  15. D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
    [PubMed]
  16. J. Janesick, Scientific charge-coupled devices, (SPIE Press, 2001).
    [CrossRef]
  17. L. Sacconi, E. Froner, and R. Antolini, M. R. Taghizadeh A. Choudhury and F. S. Pavone, "Multiphoton multifocal microscopy exploiting a diffractive optical element," Opt. Lett. 28, 1918-1920 (2003).
    [CrossRef] [PubMed]

2007

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

2004

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
[CrossRef] [PubMed]

2003

2002

B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

T. P. Padera, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte endothelial interactions," Mol. Imaging,"  1, 9-15 (2002).
[CrossRef]

2000

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

A. Egner and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A 17, 1192 (2000).
[CrossRef]

1999

1998

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655 (1998).
[CrossRef]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
[CrossRef]

1992

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

1990

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).

1989

G. S. Kino and T. R. Corle, "Confocal scanning optical microscopy," Phys. Today 42, 55-62 (1989).
[CrossRef]

1968

Antolini, R.

Bahlmann, K.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
[CrossRef] [PubMed]

Bewersdorf, J.

Blankschtein, D.

B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
[CrossRef]

Buehler, C.

Buist, A. H.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
[CrossRef]

Cody, S. H.

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

Corle, T. R.

G. S. Kino and T. R. Corle, "Confocal scanning optical microscopy," Phys. Today 42, 55-62 (1989).
[CrossRef]

Delbridge, L. M.

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).

Dudenc, R.

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

Egger, M. D.

Egner, A.

Froner, E.

Galambos, R.

Hadravsky, M.

Harris, P. J.

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

Hell, S. W.

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

A. Egner and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A 17, 1192 (2000).
[CrossRef]

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655 (1998).
[CrossRef]

Holroyd, P.

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

Huang, H.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Jahn, R.

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

Jain, R. K.

T. P. Padera, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte endothelial interactions," Mol. Imaging,"  1, 9-15 (2002).
[CrossRef]

Kim, K. H.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
[CrossRef] [PubMed]

K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
[CrossRef]

Kino, G. S.

G. S. Kino and T. R. Corle, "Confocal scanning optical microscopy," Phys. Today 42, 55-62 (1989).
[CrossRef]

Langer, R.

B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).

Lee, R. T.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Lodemann, P.

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

Majoula, I.

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

Morgan, T. O.

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

Müller, M.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
[CrossRef]

Padera, T. P.

T. P. Padera, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte endothelial interactions," Mol. Imaging,"  1, 9-15 (2002).
[CrossRef]

Petran, M.

Pick, R.

Ragan, T.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
[CrossRef] [PubMed]

Sacconi, L.

So, P. T. C.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
[CrossRef] [PubMed]

T. P. Padera, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte endothelial interactions," Mol. Imaging,"  1, 9-15 (2002).
[CrossRef]

B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).

K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
[CrossRef]

Soeling, H.-D.

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

Squier, J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
[CrossRef]

Straub, M.

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).

Sylvan, J. D.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).

Williams, D. A.

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

Yu, B. K. H. K.

B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).

Am. J. Physiol.

D. A. Williams, L. M. Delbridge, S. H. Cody, P. J. Harris, and T. O. Morgan, "Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy," Am. J. Physiol. 262, C731-42 (1992).
[PubMed]

Appl. Opt.

Eur. J. Cell Biol.

M. Straub, P. Lodemann, P. Holroyd, R. Jahn, and S. W. Hell, "Live cell imaging by multifocal multiphoton microscopy," Eur. J. Cell Biol. 79, 726-34 (2000).
[CrossRef] [PubMed]

J. Biomed. Opt.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

J. Invest. Dermatol.

B. K. H. K. Yu, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).

J. Microsc., (Oxford)

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two photon absorption microscopy using multipoint excitation," J. Microsc., (Oxford) 192, 217 (1998).
[CrossRef]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

Methods Cell Biol.

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol. 75, 23-39 (2004).
[CrossRef] [PubMed]

Mol. Imaging

T. P. Padera, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte endothelial interactions," Mol. Imaging,"  1, 9-15 (2002).
[CrossRef]

Opt. Lett.

Phys. Today

G. S. Kino and T. R. Corle, "Confocal scanning optical microscopy," Phys. Today 42, 55-62 (1989).
[CrossRef]

Rev. Mol. Biotechnol.

I. Majoula, M. Straub, R. Dudenc, S. W. Hell, and H.-D. Soeling, "Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy," Rev. Mol. Biotechnol. 82, 262-277 (2002).

Science

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).

Other

J. Bewersdorf, A. Egner, and S. W. Hell, "Multifocal Multi-Photon Microscopy" in Handbook of Biological Confocal Microscopy, J. Pawley, ed., (Springer, 2006), pp. 550-560.

J. Janesick, Scientific charge-coupled devices, (SPIE Press, 2001).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

(a). Principle of the Multifoci Multiphoton Microscope (MMM). (b) Sketch of the MMM setup. For illustration purposes, in (a) and (b) an array of only 3×3 beams is shown. (c) Still image of the foci in the focal plane. (d) Intensity profile along the axial axis. Abbreviations: Ti:Sa: Ti-sapphire Laser, MLA: Microlens Array, L: Lens, RSM: Resonant Scan Mirror, DC: Dichroic Mirror, TPB: Two-Photon Block Filter, CCD: Segmented Charged Coupled Device, Inv. M.: Inverted Microscope.

Fig. 2.
Fig. 2.

The spontaneous contraction of cardiac myocytes labeled with fluo3. The data are extracted from a 0.625 sec. movie sequence, consisting of a series of 400 images, each acquired 1.56 ms apart form each other. The number in the images indicates their place in the sequence each being 78 ms (50 images) apart. The images were smoothed in time with a Gaussian filter with four frames standard deviation. The dark current has been subtracted and the image was corrected using the flat field image.

Fig. 3.
Fig. 3.

(a). White light image of cardio myocyte shown in Fig. 2(b) Fluorescent intensity over time measured at areas 1, 2 and 3, cross sectioning the cell at 20 µm distances (a). The data indicate a wave propagation of 105 µm per second along the cell. The intensity plots are taken from the fluorescent image dataset shown in Fig. 2.

Fig. 4.
Fig. 4.

(a). Dark image of the 128×128 pix CCD camera at 640 frames/sec. The picture includes the dark and read noise of the camera. (b) Histogram of the signal of one typical segment (segment #12, framed in (a)). (c) Histogram of the mean signal values of all segments. The average dark signal of all segments is 8.7 electrons.

Tables (1)

Tables Icon

Table 1. Comparison of the presented 16 segment camera and a comparable single segment camera for two different image applications. The numbers represent the detected electrons of the system characteristics at an imaging speed of 640 frames per second.

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