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Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores

Chen, Zhixing and Wei, Lu and Zhu, Xinxin and Min, Wei (2012) Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores. Optics Express, 20 (17). pp. 18525-18536. ISSN 1094-4087. doi:10.1364/oe.20.018525.

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It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

Item Type:Article
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URLURL TypeDescription
Wei, Lu0000-0001-9170-2283
Min, Wei0000-0003-2570-3557
Additional Information:© 2012 Optical Society of America. Received 12 Apr 2012; revised 18 Jun 2012; accepted 19 Jun 2012; published 30 Jul 2012. Zhixing Chen, Lu Wei and Xinxin Zhu contributed equally to this work. We thank Ya-Ting Kao, Fang Xu, Louis Brus, Rafael Yuste, Nicholas Turro, Virginia Cornish, Darcy Peterka, Christophe Dupre and Miguel Jimenez for helpful discussions. We are grateful to Virginia Cornish for sharing lab equipments and Keith Yeager for assistance on Leica microscope. W.M. acknowledges the startup funds from Columbia University, and grant support from Kavli Institute for Brain Science.
Funding AgencyGrant Number
Columbia UniversityUNSPECIFIED
Kavli Institute for Brain ScienceUNSPECIFIED
Issue or Number:17
Classification Code:OCIS codes: (180.4315) Nonlinear microscopy; (180.2520) Fluorescence microscopy; (190.4180) Multiphoton processes; (160.4330) Nonlinear optical materials; (170.6280) Spectroscopy, fluorescence and luminescence; (170.5810) Scanning microscopy
Record Number:CaltechAUTHORS:20181119-102040297
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Official Citation:Zhixing Chen, Lu Wei, Xinxin Zhu, and Wei Min, "Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores," Opt. Express 20, 18525-18536 (2012)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91018
Deposited By: George Porter
Deposited On:19 Nov 2018 19:02
Last Modified:16 Nov 2021 03:37

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