Abstract

We demonstrate a widefield multiphoton microscope and a temporally decorrelated, multifocal, multiphoton microscope that is based on a high-efficiency array of cascaded beamsplitters. Because these microscopes use ultrashort pulse excitation over large areas of the sample, they allow efficient use of the high-average power available from modern ultrashort pulse lasers.

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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. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-4 (1997).
  3. M. Sonnleitner, G. J. Schutz, and Th Schmidt, "Imaging individual molecules by two-photon excitation," Chem. Phys. Lett. 300, 221-6 (1999).
    [CrossRef]
  4. E.H.K. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, "Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume.," Opt. Commun. 104, 223-228 (1994).
    [CrossRef]
  5. K. Konig, P. T. C. So, W. W. Mantulin, and E. Gratton, "Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes," Opt. Lett. 22, 135-6 (1997).
    [CrossRef] [PubMed]
  6. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-7 (1998).
    [CrossRef]
  7. A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microscopy 192, 217-26 (1998).
    [CrossRef]
  8. K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, "Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays," Opt. Comm. 174, 7-12 (2000).
    [CrossRef]
  9. D. N. Fittinghoff and J. A. Squier, "Time-decorrelated multifocal array for multiphoton microscopy and micromachining," Opt. Lett. 25, 1213-1215 (2000).
    [CrossRef]
  10. A. Egner and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A (Optics, Image Science and Vision) 17, 1192-201 (2000).
    [CrossRef]
  11. Heidi Dobson, "Pollen and pollen-coat lipids: chemical survey and role in pollen selection by solitary bees (Pollenkitt, Oligolecty)," PhD Dissertation, University of California, Berkeley (1985).
  12. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "I/sup 5/M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microscopy 195, 10-16 (1999).
    [CrossRef]

Other

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

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-4 (1997).

M. Sonnleitner, G. J. Schutz, and Th Schmidt, "Imaging individual molecules by two-photon excitation," Chem. Phys. Lett. 300, 221-6 (1999).
[CrossRef]

E.H.K. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, "Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume.," Opt. Commun. 104, 223-228 (1994).
[CrossRef]

K. Konig, P. T. C. So, W. W. Mantulin, and E. Gratton, "Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes," Opt. Lett. 22, 135-6 (1997).
[CrossRef] [PubMed]

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-7 (1998).
[CrossRef]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microscopy 192, 217-26 (1998).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, "Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays," Opt. Comm. 174, 7-12 (2000).
[CrossRef]

D. N. Fittinghoff and J. A. Squier, "Time-decorrelated multifocal array for multiphoton microscopy and micromachining," Opt. Lett. 25, 1213-1215 (2000).
[CrossRef]

A. Egner and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A (Optics, Image Science and Vision) 17, 1192-201 (2000).
[CrossRef]

Heidi Dobson, "Pollen and pollen-coat lipids: chemical survey and role in pollen selection by solitary bees (Pollenkitt, Oligolecty)," PhD Dissertation, University of California, Berkeley (1985).

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "I/sup 5/M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microscopy 195, 10-16 (1999).
[CrossRef]

Supplementary Material (3)

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Figures (9)

Fig. 1.
Fig. 1.

Schematic of multiphoton microscope that uses widefield illumination of the specimen.

Fig. 2.
Fig. 2.

(1.8MB) Two-photon autofluorescence video frame rate movie using a Euglena with a 100× objective. The Euglena is contracted from its normal length of ~18 µm and tumbling in place as it seeks a new direction of travel.

Fig. 3.
Fig. 3.

(1.8 MB) Two-photon autofluorescence video frame rate movie of a Euglena with a 40× objective. The Euglena contracts from its normal length of ~18 µm and tumbles as it seeks a new direction of travel.

Figure 4.
Figure 4.

Measured widefield axial point spread functions for a 100×/1.25-NA Zeiss Achroplan oil objective. The blue circles show the integrated signal as a coumarin dye cell is scanned through the focus while the red squares show the peak signal.

Figure 5.
Figure 5.

Schematic of an etalon-based temporally decorrelated, multifocal, multiphoton microscope. L1 to L4 are lenses, DM is a dichroic mirror, and M1 and M2 are mirrors, which are scanned in orthogonal directions. Please note that the three vertical dots to the right of L1 are NOT lenses. They indicate that, for clarity, the schematic is no longer showing all the beams from the etalon.

Fig. 6.
Fig. 6.

High-efficiency cascaded beamsplitters for producing a temporally decorrelated 1×8 array of beams. Lens L1 shows where the beams would enter the multifocal microscope, which is shown in its original etalon version in Fig. 4

Fig. 7.
Fig. 7.

Two-photon image of 16 foci from a cascaded-beamsplitter array. The foci are separated by ~2 µm in the horizontal direction. The entire array is asynchronously rastered by a pair of scan mirrors to produce i

Fig. 8.
Fig. 8.

White light image of a grain of pollen from Clivia Mineata. For Figure 9, a stack of sections spaced by 0.5 µm were take through the region between the vertical white lines, which is approximately 16-µm wide and 40-µm high.

Fig. 9.
Fig. 9.

(2.1 MB) 3-D rendering of a slice through a grain of Clivia Mineata pollen that was taken in 2-phton autofluorescence with the temporally decorrelated multifocal microscope.

Equations (1)

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I ( r , z ) = 2 P π ω 2 exp ( 2 r 2 ω 2 ) .

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