Widefield multiphoton and temporally decorrelated ... - OSA Publishing

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David N. Fittinghoff, Paul W. Wiseman and Jeff A. Squier ... E.H.K. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, "Nonlinear.
Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy David N. Fittinghoff, Paul W. Wiseman and Jeff A. Squier University of California at San Diego, La Jolla, Ca 92093-0339 USA [email protected]

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. 2000 Optical Society of America OCIS codes: (180.2520) Fluorescence microscopy, (180.6900) Three-dimensional microscopy

References and links 1. W. Denk, J.H. Strickler, and W.W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990). 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). 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). 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). 6. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-7 (1998). 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). 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). 9. D. N. Fittinghoff and J. A. Squier, "Time-decorrelated multifocal array for multiphoton microscopy and micromachining," Opt. Lett. 25, 1213-1215 (2000). 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). 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).

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1. Introduction

State-of-the-art ultrashort-pulse lasers used for multiphoton microscopy[1,2] have more pulse energy than is required for many biological-imaging applications. Thus, to avoid undesirable effects such as saturation or physical damage to the specimens, the input beam is attenuated from the hundreds of milliwatt or even the watt level to the milliwatt level. This is not an efficient use of expensive photons, especially when we consider that the nonlinear intensity dependence of the absorption makes multiphoton microscopy inherently confocal, which aids in efficient use of the excitation and emitted light by eliminating the need for confocal pinholes.

#23385 - $15.00 US

(C) 2000 OSA

Received August 29, 2000; Revised September 29, 2000

9 October 2000 / Vol. 7, No. 8 / OPTICS EXPRESS 273

Fig. 1 Schematic of multiphoton microscope that uses widefield illumination of the specimen.

The low average powers needed to reach high intensities with ultrashort laser pulses (