Abstract

We describe multifocal multiphoton microscopy giving images without laser scanning. A multitude of 8 × 8 laser beams is focused into a sample yielding two-photon excitation in a plane. The focal spots are arranged in a rectangular array with close spacing between individual points (≈0.5 µm). The fluorescence emission from the sample is recorded with a CCD camera, but, owing to the close distance between the beams, they can no longer be regarded as individual points but rather as an illumination of the plane that is covered by the array of focal points. The axial sectioning capability is comparable with an ordinary single-beam two-photon microscope. Interference between the beams that could compromise the axial sectioning capability does not occur in our setup owing to small temporal delays between the individual beams. The axial sectioning capability of the setup is discussed in detail by means of the step response in which the foci are scanned axially into a uniformly fluorescent medium.

© 2005 Optical Society of America

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References

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  1. J. B. Pawley, ed. Handbook of Biological Confocal Microscopy (Plenum, New York, 1995).
  2. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,”Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  3. A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
    [CrossRef]
  4. J. Bewersdorf, R. Pick, S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23, 655–657 (1998).
    [CrossRef]
  5. A. Egner, S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17, 1192–1201 (2000).
    [CrossRef]
  6. S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
    [CrossRef]
  7. T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
    [CrossRef] [PubMed]

2001 (1)

T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
[CrossRef] [PubMed]

2000 (1)

1998 (2)

A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
[CrossRef]

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

1993 (1)

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[CrossRef]

1990 (1)

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

Andresen, P.

T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
[CrossRef] [PubMed]

Bewersdorf, J.

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
[CrossRef]

Cremer, C.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[CrossRef]

Denk, W.

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

Egner, A.

Fricke, M.

T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
[CrossRef] [PubMed]

Hell, S.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[CrossRef]

Hell, S. W.

Hellweg, D.

T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
[CrossRef] [PubMed]

Müller, M.

A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
[CrossRef]

Nielsen, T.

T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
[CrossRef] [PubMed]

Pick, R.

Reiner, G.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[CrossRef]

Squier, J.

A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
[CrossRef]

Stelzer, E. H. K.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[CrossRef]

Strickler, J. H.

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

Webb, W. W.

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

J. Microsc. (3)

A. H. Buist, M. Müller, J. Squier, G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192, 217–226 (1998).
[CrossRef]

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, “Aberations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169, 391–405 (1993).
[CrossRef]

T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, “High efficiency beam splitter for multifocal multiphoton microscopy,” J. Microsc. 201, 368–376 (2001).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (1)

Opt. Lett. (1)

Science (1)

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

Other (1)

J. B. Pawley, ed. Handbook of Biological Confocal Microscopy (Plenum, New York, 1995).

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Figures (8)

Fig. 1
Fig. 1

Experimental setup.

Fig. 2
Fig. 2

Principle of the beam splitter.

Fig. 3
Fig. 3

Fluorescence from a homogeneous solution of Rhodamine 6G in water. The arrows indicate the lines along which the profiles of Fig. 4 were taken.

Fig. 4
Fig. 4

Intensity profiles of the image displayed in Fig. 3. The profiles belong to the lines identified by the arrows and symbols in Fig. 3. From this, the uniformity of the illumination can be seen. For comparison, the profile of a single beam is also included in the plot.

Fig. 5
Fig. 5

Image of fluorescent latex beads.

Fig. 6
Fig. 6

Fluorescence image of yeast cells stained with Rhodamine 6G and a bright-field image of the cells. The region that is covered by the laser foci is indicated by the white rectangle in the bright-field image.

Fig. 7
Fig. 7

Response curves that are obtained if the foci are scanned axially into a fluorescent sea: all beams (solid curve), single beam (dashed curve), and theoretical limit (dashed–dotted curve).

Fig. 8
Fig. 8

Response curves that are obtained if the foci are scanned axially into a fluorescent sea: multiple foci covering an area of 4 µm × 4 µm (solid curve) and a single focused beam with an equivalent area of the focus (dashed curve). Obviously the multibeam approach results in much better axial resolution than a single beam.

Equations (2)

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I ( z ) = A π [ π 2 + arctan ( z z 0 z R ) + 1 z R z z 0 + z z 0 z R ] ,
z R = 1.169 ( n λ N . A . 2 ) = 0.86 µ m ,

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