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Proceedings Paper

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging
Author(s): Lu Wei; Xinxin Zhu; Zhixing Chen; Wei Min
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Paper Abstract

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

Paper Details

Date Published: 28 February 2014
PDF: 8 pages
Proc. SPIE 8948, Multiphoton Microscopy in the Biomedical Sciences XIV, 894825 (28 February 2014); doi: 10.1117/12.2038753
Show Author Affiliations
Lu Wei, Columbia Univ. (United States)
Xinxin Zhu, Columbia Univ. (United States)
Zhixing Chen, Columbia Univ. (United States)
Wei Min, Columbia Univ. (United States)
Kavli Institute for Brain Science (United States)


Published in SPIE Proceedings Vol. 8948:
Multiphoton Microscopy in the Biomedical Sciences XIV
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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