Abstract

A brief overview of nonlinear microscopy in biomedicine is presented. Some of the main results of the contributions of the Focus Issue are also briefly discussed.

© Optical Society of America

1. Introduction

It has been almost a decade since the potential of using nonlinear optical effect for high resolution microscopy imaging was realized [1]. Many of the basic technical issues related to this new technology have been resolved. The resolution of multiphoton microscopes has been measured and verified against theoretical predictions [2,3]. The quantum yields of fluorescent probes suitable for multiphoton microscopy have been determined and new probes optimized for this application are being synthesized [4,5]. In depth comparison between two-photon and confocal microscopy has been performed [6].

In addition, two-photon microscopy has found novel applications in many areas of biology, biophysics, and medicine. The efficiency of two-photon excitation to discriminate against background fluorescence has allowed the development of two-photon single molecule spectroscopy and two-photon fluorescence correlation spectroscopy [7, 8]. The combination of two-photon excitation with fluorescence spectroscopy enables in vivo monitoring of intracellular biochemistry [9,10,11]. The low tissue absorbancy of infrared light and the non-invasive nature of two-photon excitation has facilitated major advances in embryology studies and noninvasive tissue diagnosis [12–15]. Also see papers by Mohler and White and So et al. in this issue. A combination of confocal reflected light and two-photon fluorescence imaging has been shown to provide complementary structural information in tissues [16, 17, see also Masters in this issue]. Realizing that three- and higher-photon excitation in microscope is useful for imaging deep UV chromophores, three- and high photon microscopy has been developed [18,19,20]. The unique ability of two-photon microscopy to effect chemical reaction in a subfemtoliter volume has been utilized to activate caged compounds with exquisite spatial precision [21,22]. Realizing that the data rate of a typical scanning microscope is too slow for clinical applications or to observe fast kinetics in cells, video rate two-photon microscope systems have been developed [23,24]. The development of multiphoton imaging systems based on a lower cost, picosecond pulse laser or cw lasers may allow wider adoption of this new technology [25]. Finally, realizing that fluorescent labeling of a specimen is sometimes undesirable, nonlinear microscopy based on second and third harmonic light generation has been developed [26]. Also see Squier et al. in this issue.

The technology development in the field of nonlinear microscopy has been exciting. The pace of this innovation is expected to continue but may be at a slower rate as multi-photon imaging reaches maturity. Recently, we have observed that the number of biological and medical researchers adopting multiphoton microscopy as a routine laboratory tool has been increasing rapidly. Hopefully, the next decade will be a time in which the development practice of multiphoton microscopy will come to fruition bringing new scientific insights in biology and improving the practice to medical procedures.

References and links

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

2. C. J. R. Sheppard and M. Gu, M., “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

3. S. W. Hell, S. Lindek, and E. H. K. Stelzer, “Enhancing the axial resolution in the far-field light microscopy: two-photon 4-Pi confocal fluorescence microscopy,” J. Mod. Opt. 41, 675–81 (1994). [CrossRef]  

4. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biologicalnonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996). [CrossRef]   [PubMed]  

5. M. Albota, D. Beljonne, J. L. Bredas, J. E. Ehrlich, J. Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D., McCord-Maughon, J. W. Perry, H. Rockel, M. Rumi, G. Subramaniam, W. W. Webb, X. L. Wu, and C. Xu, “Design of organic molecules with large two-photon absorption cross sections,” Science 281, 1653–1656 (1998). [CrossRef]   [PubMed]  

6. V. E. Centonze and J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys J. 75, 2015–2024 (1998). [CrossRef]   [PubMed]  

7. J. Mertz, C. Xu, and W. W. Webb, “Single-molecule detection by two-photon excited fluorescence”, Opt. Lett. , 20, 2532–2534 (1996). [CrossRef]  

8. K. M. Berland, P. T. C. So, C. Y. Dong, W. W. Mantulin, and E. Gratton, “Scanning Two-Photon Fluctuation Correlation Spectroscopy: Particle Counting Measurement for Detection of Molecular Aggregation,” Biophys. J. 71, 410–420 (1996). [CrossRef]   [PubMed]  

9. T. French, P. T. C. So, D. J. Weaver Jr, T. Coelho-Sampaio, E. Gratton E, E. W. Voss Jr, and J. Carrero, “Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing,” J. Microsc. 185, 339–353 (1997). [CrossRef]   [PubMed]  

10. J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191, 39 (1998). [CrossRef]  

11. W. M. Yu, P. T. C. So, T. French, and E. Gratton, “Fluorescence Polarization of Cell Membrane - A Two- Photon Scanning Microscopy Approach,” Biophys. J. 70, 626–636 (1996). [CrossRef]   [PubMed]  

12. R. G. Summers, D. W. Piston, K. M. Harris, and J. B. Morrill, “The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry,” Dev. Biol. 175, 177–183 (1996). [CrossRef]   [PubMed]  

13. W. A. Wohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin JD, and J. G. White, “Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis,” Curr. Biol. 8, 1087–1090 (1998). [CrossRef]  

14. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Micros. 178, 20–27, (1995). [CrossRef]  

15. B. R. Masters, P. T. C. So, and E. Gratton, “Multi-Photon Excitation Fluorescence Microscopy and Spectroscopy of In Vivo Human Skin,” Biophys. J. 72, 2405–2412 (1997). [CrossRef]   [PubMed]  

16. K. H. Kim, P. T. C. So, I. E. Kochevar, B. R. Masters, and E. Gratton, “Two-Photon Fluorescence and Confocal Reflected Light Imaging of Thick Tissue Structures,” SPIE 3260, 46–57 (1998). [CrossRef]  

17. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest Dermatol. 104, 946–952 (1995). [CrossRef]   [PubMed]  

18. J. R. Lakowicz, I. Gryczynski, H. Malak, M. Schrader, P. Engelhardt, H. Kano, and S. W. Hell, “Time-resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-photon excitation,” Biophys. J. 72, 567–78 (1997). [CrossRef]   [PubMed]  

19. D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker, and A. I. Ferguson, “Multi-photon excitation imaging with an all-solid-state laser,” Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 2678, 38–49 (1996).

20. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997). [CrossRef]   [PubMed]  

21. K. Svoboda, W. Denk W, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385,161–165 (1997). [CrossRef]   [PubMed]  

22. P. Lipp and E. Niggli, “Fundamental calcium release events revealed by two-photon excitation photolysis of caged calcium in Guinea-pig cardiac myocytes,” J. Physiol. (London) 508, 801–809 (1998). [CrossRef]  

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

24. J. Bewersdorf, R. Pick, and S. W. Hell, “Mulitfocal multiphoton microscopy,” Opt. Lett. 23, 655 (1998). [CrossRef]  

25. M. J. Booth and S. W. Hell, “Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line,” J. Microsc. 190, 298–304 (1998). [CrossRef]   [PubMed]  

26. M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998). [CrossRef]   [PubMed]  

References

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  1. W. Denk, J. H. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  2. C. J. R., Sheppard, and M. Gu, M., "Image formation in two-photon fluorescence microscopy," Optik 86, 104-106 (1990).
  3. S. W. Hell, S. Lindek and E. H. K. Stelzer, "Enhancing the axial resolution in the far-field light microscopy: two-photon 4-Pi confocal fluorescence microscopy," J. Mod. Opt. 41, 675-81 (1994).
    [CrossRef]
  4. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biologicalnonlinear microscopy," Proc. Natl. Acad. Sci. USA 93, 10763-10768 (1996).
    [CrossRef] [PubMed]
  5. M. Albota, D. Beljonne, J. L. Bredas, J. E. Ehrlich, J. Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D., McCord-Maughon, J. W. Perry, H. Rockel, M. Rumi, G. Subramaniam, W. W. Webb, X. L. Wu, C. Xu, "Design of organic molecules with large two-photon absorption cross sections," Science 281, 1653-1656 (1998).
    [CrossRef] [PubMed]
  6. V. E. Centonze, J. G. White, "Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging," Biophys J. 75, 2015-2024 (1998).
    [CrossRef] [PubMed]
  7. J. Mertz, C. Xu and W. W. Webb, "Single-molecule detection by two-photon excited fluorescence", Opt. Lett., 20, 2532-2534 (1996).
    [CrossRef]
  8. K. M. Berland, P. T. C. So, C. Y. Dong, W. W. Mantulin and E. Gratton, "Scanning Two-Photon Fluctuation Correlation Spectroscopy: Particle Counting Measurement for Detection of Molecular Aggregation," Biophys. J. 71, 410-420 (1996).
    [CrossRef] [PubMed]
  9. T. French, P. T. C. So, D. J. Weaver Jr, T. Coelho-Sampaio, E. Gratton E, E. W.Voss Jr. and J. Carrero, "Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing," J. Microsc. 185, 339-353 (1997).
    [CrossRef] [PubMed]
  10. J. Sytsma, J. M. Vroom, C. J. De Grauw, H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39 (1998).
    [CrossRef]
  11. W. M. Yu, P. T. C. So, T. French and E. Gratton, "Fluorescence Polarization of Cell Membrane - A Two- Photon Scanning Microscopy Approach," Biophys. J. 70, 626-636 (1996).
    [CrossRef] [PubMed]
  12. R. G. Summers, D. W. Piston, K. M. Harris and J. B. Morrill, "The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry," Dev. Biol. 175, 177-183 (1996).
    [CrossRef] [PubMed]
  13. W. A. Wohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin JD and J. G. White, "Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis," Curr. Biol. 8, 1087-1090 (1998).
    [CrossRef]
  14. W. Piston, B. R. Masters, and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Micros. 178, 20-27, (1995).
    [CrossRef]
  15. B. R. Masters, P. T. C. So, and E. Gratton, "Multi-Photon Excitation Fluorescence Microscopy and Spectroscopy of In Vivo Human Skin," Biophys. J. 72, 2405-2412 (1997).
    [CrossRef] [PubMed]
  16. K. H. Kim, P. T. C. So, I. E. Kochevar, B. R. Masters and E. Gratton, "Two-Photon Fluorescence and Confocal Reflected Light Imaging of Thick Tissue Structures," SPIE 3260, 46-57 (1998).
    [CrossRef]
  17. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb and R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Invest Dermatol. 104, 946-952 (1995).
    [CrossRef] [PubMed]
  18. J. R. Lakowicz, I. Gryczynski, H. Malak, M. Schrader, P. Engelhardt, H. Kano and S. W. Hell, "Time- resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-photon excitation," Biophys. J. 72, 567-78 (1997).
    [CrossRef] [PubMed]
  19. D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker and A. I. Ferguson, "Multi-photon excitation imaging with an all-solid-state laser," Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 2678, 38-49 (1996).
  20. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel and W. W. Webb, "Measuring serotonin distribution in live cells with three-photon excitation," Science 275, 530-532 (1997).
    [CrossRef] [PubMed]
  21. K. Svoboda, W. Denk W, D. Kleinfeld and D. W. Tank, "In vivo dendritic calcium dynamics in neocortical pyramidal neurons," Nature 385,161-165 (1997).
    [CrossRef] [PubMed]
  22. P. Lipp and E. Niggli, "Fundamental calcium release events revealed by two-photon excitation photolysis of caged calcium in Guinea-pig cardiac myocytes," J. Physiol. (London) 508, 801-809 (1998).
    [CrossRef]
  23. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade and B. Athey, "Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system," J. Microsc. 181, 253 (1996).
    [CrossRef] [PubMed]
  24. J. Bewersdorf, R. Pick, and S. W. Hell, "Mulitfocal multiphoton microscopy," Opt. Lett. 23, 655 (1998).
    [CrossRef]
  25. M. J. Booth and S. W. Hell, "Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line," J. Microsc. 190, 298-304 (1998).
    [CrossRef] [PubMed]
  26. M. Muller, J. Squier, K. R. Wilson and G. J. Brakenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
    [CrossRef] [PubMed]

Other (26)

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

C. J. R., Sheppard, and M. Gu, M., "Image formation in two-photon fluorescence microscopy," Optik 86, 104-106 (1990).

S. W. Hell, S. Lindek and E. H. K. Stelzer, "Enhancing the axial resolution in the far-field light microscopy: two-photon 4-Pi confocal fluorescence microscopy," J. Mod. Opt. 41, 675-81 (1994).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biologicalnonlinear microscopy," Proc. Natl. Acad. Sci. USA 93, 10763-10768 (1996).
[CrossRef] [PubMed]

M. Albota, D. Beljonne, J. L. Bredas, J. E. Ehrlich, J. Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D., McCord-Maughon, J. W. Perry, H. Rockel, M. Rumi, G. Subramaniam, W. W. Webb, X. L. Wu, C. Xu, "Design of organic molecules with large two-photon absorption cross sections," Science 281, 1653-1656 (1998).
[CrossRef] [PubMed]

V. E. Centonze, J. G. White, "Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging," Biophys J. 75, 2015-2024 (1998).
[CrossRef] [PubMed]

J. Mertz, C. Xu and W. W. Webb, "Single-molecule detection by two-photon excited fluorescence", Opt. Lett., 20, 2532-2534 (1996).
[CrossRef]

K. M. Berland, P. T. C. So, C. Y. Dong, W. W. Mantulin and E. Gratton, "Scanning Two-Photon Fluctuation Correlation Spectroscopy: Particle Counting Measurement for Detection of Molecular Aggregation," Biophys. J. 71, 410-420 (1996).
[CrossRef] [PubMed]

T. French, P. T. C. So, D. J. Weaver Jr, T. Coelho-Sampaio, E. Gratton E, E. W.Voss Jr. and J. Carrero, "Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing," J. Microsc. 185, 339-353 (1997).
[CrossRef] [PubMed]

J. Sytsma, J. M. Vroom, C. J. De Grauw, H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39 (1998).
[CrossRef]

W. M. Yu, P. T. C. So, T. French and E. Gratton, "Fluorescence Polarization of Cell Membrane - A Two- Photon Scanning Microscopy Approach," Biophys. J. 70, 626-636 (1996).
[CrossRef] [PubMed]

R. G. Summers, D. W. Piston, K. M. Harris and J. B. Morrill, "The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry," Dev. Biol. 175, 177-183 (1996).
[CrossRef] [PubMed]

W. A. Wohler, J. S. Simske, E. M. Williams-Masson, J. D. Hardin JD and J. G. White, "Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis," Curr. Biol. 8, 1087-1090 (1998).
[CrossRef]

W. Piston, B. R. Masters, and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Micros. 178, 20-27, (1995).
[CrossRef]

B. R. Masters, P. T. C. So, and E. Gratton, "Multi-Photon Excitation Fluorescence Microscopy and Spectroscopy of In Vivo Human Skin," Biophys. J. 72, 2405-2412 (1997).
[CrossRef] [PubMed]

K. H. Kim, P. T. C. So, I. E. Kochevar, B. R. Masters and E. Gratton, "Two-Photon Fluorescence and Confocal Reflected Light Imaging of Thick Tissue Structures," SPIE 3260, 46-57 (1998).
[CrossRef]

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb and R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Invest Dermatol. 104, 946-952 (1995).
[CrossRef] [PubMed]

J. R. Lakowicz, I. Gryczynski, H. Malak, M. Schrader, P. Engelhardt, H. Kano and S. W. Hell, "Time- resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-photon excitation," Biophys. J. 72, 567-78 (1997).
[CrossRef] [PubMed]

D. L. Wokosin, V. E. Centonze, J. G. White, S. N. Hird, S. Sepsenwol, G. P. A. Malcolm, G. T. Maker and A. I. Ferguson, "Multi-photon excitation imaging with an all-solid-state laser," Proc. of Optical Diagnostics of Living Cells and Biofluids, SPIE 2678, 38-49 (1996).

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel and W. W. Webb, "Measuring serotonin distribution in live cells with three-photon excitation," Science 275, 530-532 (1997).
[CrossRef] [PubMed]

K. Svoboda, W. Denk W, D. Kleinfeld and D. W. Tank, "In vivo dendritic calcium dynamics in neocortical pyramidal neurons," Nature 385,161-165 (1997).
[CrossRef] [PubMed]

P. Lipp and E. Niggli, "Fundamental calcium release events revealed by two-photon excitation photolysis of caged calcium in Guinea-pig cardiac myocytes," J. Physiol. (London) 508, 801-809 (1998).
[CrossRef]

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

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

M. J. Booth and S. W. Hell, "Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line," J. Microsc. 190, 298-304 (1998).
[CrossRef] [PubMed]

M. Muller, J. Squier, K. R. Wilson and G. J. Brakenhoff, "3D microscopy of transparent objects using third-harmonic generation," J. Microsc. 191, 266-274 (1998).
[CrossRef] [PubMed]

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