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Deep image with non-degenerated two-photon excitation (Conference Presentation)
Author(s): Mu-Han Yang; Sanaz Sadegh; Payam A. Saisan; Christopher G. L. Ferri; Anna Devor; Yeshaiahu Fainman
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Paper Abstract

In non-degenerate 2-photon excitation (ND-2PE) microscopy, a fluorophore simultaneously absorbs two photons of different energies. We performed a ND-2PE study of fluorescent proteins and synthetic dyes (eg.eGFP, FITC, and etc.) continuously varying energies and numbers of both photons to create two-dimensional map of fluorescence landscapes. By using the best photon energy combination from our two-dimensional map, we found an increase in detected fluorescent image brightness with ND-2PE as we imaged cortical neurons labeled with enhanced green fluorescent protein (eGFP). It should be noted that the photons corresponding to longer wavelength will penetrate deeper into the tissue at reduced scattering. Additionally, using non-overlapping spatial modes carrying the photons at different energies will significantly reduce out of focus fluorescence from the large number of low energy photons, and by a proper choice of the number of high energy photons the ND-2PE fluorescence can be obtained from deep tissue. Experimentally, we strategically displaced two laser beams until they reached the sample plane such that the unwanted background in the excitation beam path was suppressed. In contrast, these two pump beams were well overlapped at focus which still produced sufficient number fluorescence photons for detection. In our experiment, the temporal alignment was achieved with optical delay line in the optical path of IR beam. With this technique we demonstrated experimentally that ND-2PE with side-by-side beams provided a better signal to background ratio in the scattering phantom as compared with D-2PE. The excitation volume of ND-2PE with side-by-side beam was also investigated and determined to be comparable in size with that of the D-2PE.

Paper Details

Date Published: 17 September 2018
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Proc. SPIE 10753, Ultrafast Nonlinear Imaging and Spectroscopy VI, 107530E (17 September 2018); doi: 10.1117/12.2320384
Show Author Affiliations
Mu-Han Yang, Univ. of California, San Diego (United States)
Sanaz Sadegh, Univ. of California, San Diego (United States)
Payam A. Saisan, Univ. of California, San Diego (United States)
Christopher G. L. Ferri, Univ. of California, San Diego (United States)
Anna Devor, Univ. of California, San Diego (United States)
Yeshaiahu Fainman, Univ. of California, San Diego (United States)


Published in SPIE Proceedings Vol. 10753:
Ultrafast Nonlinear Imaging and Spectroscopy VI
Zhiwen Liu; Demetri Psaltis; Kebin Shi, Editor(s)

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