Deep tissue two-photon microscopy

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  • A Corrigendum to this article was published on 01 March 2006

Abstract

With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional—including confocal—fluorescence microscopy. Nonlinear optical microscopy, in particular two photon–excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

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Figure 1: Nonlinear optical microscopy.
Figure 2: Signal generation and fluorescence collection in clear tissue (no scatter) and in scattering tissue (scatter).
Figure 3: Beam size adjustment relative to the objective's back aperture.
Figure 4: In vivo two-photon imaging in the intact neocortex.

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Acknowledgements

We thank T. Kuner, G. Augustine and G. Feng for providing the Clomeleon mouse and W. Göbel for help with numerical calculations.

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Correspondence to Fritjof Helmchen or Winfried Denk.

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Competing interests

W.D. is one of the authors on a patent on two-photon microscopy (US Patent 5034613).

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Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nat Methods 2, 932–940 (2005) doi:10.1038/nmeth818

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