Abstract

We have developed a high-speed two-photon microscope with submicrometer resolution in real time. The imaging speed improvement of this system is obtained by the use of a high-speed polygonal mirror scanner. The maximum achievable scanning rate is 40 µs/line, which is approximately 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real time by an intensified CCD camera. Using this instrument, we have resolved cellular architecture in three dimensions and have monitored the movements of protozoas. More important, photodamage to biological specimens during video-rate imaging can be minimized with two-photon excitation as compared with other one-photon modalities.

© 1999 Optical Society of America

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  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. W. A. Mohler, J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” Biotechniques 24, 1006–1010 (1998).
    [PubMed]
  3. B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
    [CrossRef] [PubMed]
  4. K. Svoboda, D. W. Tank, W. Denk, “Direct measurement of coupling between dendritic spines and shafts,” Science 272, 716–719 (1996).
    [CrossRef] [PubMed]
  5. B. R. Masters, P. T. C. So, E. Gratton, “Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
    [CrossRef] [PubMed]
  6. P. T. So, H. K. Kim, I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Exp. 3, 339–350 (1998).
    [CrossRef]
  7. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
    [CrossRef] [PubMed]
  8. J. B. Guild, W. W. Webb, “Line scanning microscopy with two-photon fluorescence excitation,” Biophys. J. 68, 290a (1995).
  9. J. Bewersdorf, R. Pick, S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23, 655–657 (1998).
    [CrossRef]
  10. A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
    [CrossRef]
  11. M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
    [CrossRef]

1998 (4)

W. A. Mohler, J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” Biotechniques 24, 1006–1010 (1998).
[PubMed]

P. T. So, H. K. Kim, I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Exp. 3, 339–350 (1998).
[CrossRef]

J. Bewersdorf, R. Pick, S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23, 655–657 (1998).
[CrossRef]

A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
[CrossRef]

1997 (1)

B. R. Masters, P. T. C. So, E. Gratton, “Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef] [PubMed]

1996 (3)

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

K. Svoboda, D. W. Tank, W. Denk, “Direct measurement of coupling between dendritic spines and shafts,” Science 272, 716–719 (1996).
[CrossRef] [PubMed]

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

1995 (2)

J. B. Guild, W. W. Webb, “Line scanning microscopy with two-photon fluorescence excitation,” Biophys. J. 68, 290a (1995).

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Anderson, R. R.

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

Athey, B.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Bennett, B. D.

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

Bewersdorf, J.

Bliton, A. C.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Brakenhoff, G. J.

A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
[CrossRef]

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Buist, A. H.

A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
[CrossRef]

Denk, W.

K. Svoboda, D. W. Tank, W. Denk, “Direct measurement of coupling between dendritic spines and shafts,” Science 272, 716–719 (1996).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Esterowitz, D.

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

Gratton, E.

B. R. Masters, P. T. C. So, E. Gratton, “Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef] [PubMed]

Grossman, M.

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

Guild, J. B.

J. B. Guild, W. W. Webb, “Line scanning microscopy with two-photon fluorescence excitation,” Biophys. J. 68, 290a (1995).

Hell, S. W.

Jetton, T. L.

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

Kim, H. K.

P. T. So, H. K. Kim, I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Exp. 3, 339–350 (1998).
[CrossRef]

Kochevar, I. E.

P. T. So, H. K. Kim, I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Exp. 3, 339–350 (1998).
[CrossRef]

Magnuson, M. A.

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

Masters, B. R.

B. R. Masters, P. T. C. So, E. Gratton, “Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef] [PubMed]

Mohler, W. A.

W. A. Mohler, J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” Biotechniques 24, 1006–1010 (1998).
[PubMed]

Muller, M.

A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
[CrossRef]

Norris, T.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Pick, R.

Piston, P. D.

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

Raijadhyaksha, M.

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

So, P. T.

P. T. So, H. K. Kim, I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Exp. 3, 339–350 (1998).
[CrossRef]

So, P. T. C.

B. R. Masters, P. T. C. So, E. Gratton, “Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef] [PubMed]

Squier, J.

A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
[CrossRef]

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Svoboda, K.

K. Svoboda, D. W. Tank, W. Denk, “Direct measurement of coupling between dendritic spines and shafts,” Science 272, 716–719 (1996).
[CrossRef] [PubMed]

Tank, D. W.

K. Svoboda, D. W. Tank, W. Denk, “Direct measurement of coupling between dendritic spines and shafts,” Science 272, 716–719 (1996).
[CrossRef] [PubMed]

Wade, W. H.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Webb, R. H.

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

Webb, W. W.

J. B. Guild, W. W. Webb, “Line scanning microscopy with two-photon fluorescence excitation,” Biophys. J. 68, 290a (1995).

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

White, J. G.

W. A. Mohler, J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” Biotechniques 24, 1006–1010 (1998).
[PubMed]

Ying, G.

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

Biophys. J. (2)

B. R. Masters, P. T. C. So, E. Gratton, “Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef] [PubMed]

J. B. Guild, W. W. Webb, “Line scanning microscopy with two-photon fluorescence excitation,” Biophys. J. 68, 290a (1995).

Biotechniques (1)

W. A. Mohler, J. G. White, “Stereo-4-D reconstruction and animation from living fluorescent specimens,” Biotechniques 24, 1006–1010 (1998).
[PubMed]

J Microsc. (1)

A. H. Buist, M. Muller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J Microsc. 192, 217–26 (1998).
[CrossRef]

J. Biol. Chem. (1)

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson, P. D. Piston, “Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets,” J. Biol. Chem. 271, 3647–3651 (1996).
[CrossRef] [PubMed]

J. Investigative Dermatol. (1)

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Investigative Dermatol. 6, 946–952 (1995).
[CrossRef]

J. Microsc. (1)

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade, B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181, 253–259 (1996).
[CrossRef] [PubMed]

Opt. Exp. (1)

P. T. So, H. K. Kim, I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Exp. 3, 339–350 (1998).
[CrossRef]

Opt. Lett. (1)

Science (2)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

K. Svoboda, D. W. Tank, W. Denk, “Direct measurement of coupling between dendritic spines and shafts,” Science 272, 716–719 (1996).
[CrossRef] [PubMed]

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Figures (6)

Fig. 1
Fig. 1

Schematic of the high-speed, two-photon scanning microscope.

Fig. 2
Fig. 2

Timing diagram for the synchronization of actuators and sensor. The signal from the photodiode is interpreted as an x-axis end-of-line signal. Y-axis scanner changes its position synchronously with this end-of-line signal. After 256 lines are scanned (one plane: 256 × 256 pixels), the z-axis piezoelectric objective translator steps to the next plane. CCD camera also runs synchronously with the piezoelectric translator.

Fig. 3
Fig. 3

(a)–(c) Time series of a 100-µm piezoinduced linear movement of 2-µm, yellow-green spheres. Three typical images of a movie of 100 frames are depicted. (d) Accumulative image over the same time course as in (a).

Fig. 4
Fig. 4

Two-photon, 3-D resolved images of mitochondria distribution in mouse fibroblast cells as revealed with dihydrorhodamine labeling. Left panel shows a typical two-dimensional slice. Right panel shows the 3-D reconstruction.

Fig. 5
Fig. 5

Stroboscopically (11 frames/s) recorded movements of Calcein-AM-labeled blepharisma in an aqueous environment. Images were taken with the 25× water-immersion objective.

Fig. 6
Fig. 6

Time-lapse sequences of euglena’s movement. (a) Imaged with a wide-field fluorescence video microscope. Euglena was paralyzed within 3 s. (b) Imaged with a two-photon video-rate microscope. No photodamage was observed. The frame size is 62 µm.

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