We study and demonstrate the technique of simultaneous spatial and temporal focusing of femtosecond pulses, with the aim to improve the signal-to-background ratio in multiphoton imaging. This concept is realized by spatially separating spectral components of pulses into a “rainbow beam” and recombining these components only at the spatial focus of the objective lens. Thus, temporal pulse width becomes a function of distance, with the shortest pulse width confined to the spatial focus. We developed analytical expressions to describe this method and experimentally demonstrated the feasibility. The concept of simultaneous spatial and temporal focusing of femtosecond pulses has the great potential to significantly reduce the background excitation in multiphoton microscopy, which fundamentally limits the imaging depth in highly scattering biological specimens.

© 2005 Optical Society of America

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J. Microsc.

• 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, 253-259 (1996). [CrossRef] [PubMed]

J. Neuroscience Methods

• W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Strawbridge, D. W. Tank, and R. Yuste, �??Anatomical and functional imaging of neurons using 2-photon laser microscopy,�?? J. Neuroscience Methods, 54, 161-162 (1994). [CrossRef]

J. Opt. Soc. Am.

• O. E. Martinez, �??Grating and prism compressor in the case of finite beam size,�?? J. Opt. Soc. Am. 3, 929-934, 1986. [CrossRef]

Nature Biotechnol.

• J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, �??Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,�?? Nature Biotechnol. 17, 763-767 (1999). [CrossRef]

Opt. Express

• During our research work of temporal focusing, we become aware, through the reviewing process, that a manuscript was submitted on a similar concept for wide field imaging. D. Oron, E. Tal and Y. Silberberg, �??Scanningless depth-resolved microscopy,�?? Opt. Express 13, 1468-1476 (2005), <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-13-5-1468.">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-13-5-1468.</a> [CrossRef] [PubMed]

Opt. Lett.

• C. Xu, J. Guild, W. W. Webb and W. Denk, �??Determination of absolute two-photon cross sections by in situ second order autocorrelations,�?? Opt. Lett. 20, 2372-2374 (1995). [CrossRef] [PubMed]
• P. Theer, M. T. Hasan, and W. Denk, "Two-photon imaging to a depth 1000um in living brains by use of Ti:Al2O3 regenerative amplifier,�?? Opt. Lett. 28, 1022-1024 (2003). [CrossRef] [PubMed]

Proc. Natl. Acad. Sci.

• W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, �??Live tissue intrinsic emission microscopy using multi-photon intrinsic fluorescence and second harmonic generation,�?? Proc. Natl. Acad. Sci. 100, 7075-7080 (2003). [CrossRef] [PubMed]

Other

• H. A. Haus, Waves and fields in optoelectronics, (Prentice-Hall, Englewood Cliffs, NJ, 1984), Chap 4.

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