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Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

Wei, Lu and Zhu, Xinxin and Chen, Zhixing and Min, Wei (2014) Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging. In: Multiphoton Microscopy in the Biomedical Sciences XIV. Proceedings of SPIE. No.8948. Society of Photo-optical Instrumentation Engineers (SPIE) , Bellingham, WA, Art. No. 894825. ISBN 9780819498618.

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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.

Item Type:Book Section
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URLURL TypeDescription
Wei, Lu0000-0001-9170-2283
Min, Wei0000-0003-2570-3557
Additional Information:© 2014 Society of Photo-Optical Instrumentation Engineers (SPIE).
Subject Keywords:Nonlinear microscopy, multiphoton microscopy, imaging-depth limit, real population transfer, deep-tissue imaging
Series Name:Proceedings of SPIE
Issue or Number:8948
Record Number:CaltechAUTHORS:20180608-133651608
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Official Citation:Lu Wei, Xinxin Zhu, Zhixing Chen, Wei Min, "Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging", Proc. SPIE 8948, Multiphoton Microscopy in the Biomedical Sciences XIV, 894825 (28 February 2014); doi: 10.1117/12.2038753;
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:86928
Deposited By: George Porter
Deposited On:08 Jun 2018 22:31
Last Modified:15 Nov 2021 20:43

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