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Proceedings Paper

Multi-color femtosecond source for simultaneous excitation of multiple fluorescent proteins in two-photon fluorescence microscopy
Author(s): Ke Wang; Tzu-Ming Liu; Juwell Wu; Nicholas G. Horton; Charles P. Lin; Chris Xu
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Paper Abstract

Simultaneous imaging of cells expressing multiple fluorescent proteins (FPs) is of particular interest in applications such as mapping neural circuits, tracking multiple immune cell populations, etc. To visualize both in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissues, two-photon fluorescence microscopy (2PM) is a powerful tool that has found wide applications. However, simultaneous imaging of multiple FPs with 2PM is greatly hampered by the lack of proper ultrafast lasers offering multi-color femtosecond pulses, each targeting the two-photon absorption peak of a different FP. Here we demonstrate simultaneous two-photon fluorescence excitation of RFP, YFP, and CFP in human melanoma cells engineered to express a “rainbow” pallet of colors, using a novel fiber-based source with energetic, three-color femtosecond pulses. The three-color pulses, centered at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation of the 1550 nm pump laser and SHG of the solitons at 1728 nm and 1900 nm generated through soliton self-frequency shift (SSFS) of the pump laser in a large-mode-area (LMA) fiber. The resulting wavelengths are well matched to the two-photon absorption peaks of the three FPs for efficient excitation. Our results demonstrate that multi-color femtosecond pulse generation using SSFS and a turn-key, fiber-based femtosecond laser can fulfill the requirements for simultaneous imaging of multiple FPs in 2PM, opening new opportunities for a wide range of biological applications where non-invasive, high-resolution imaging of multiple fluorescent indicators is required.

Paper Details

Date Published: 22 February 2013
PDF: 6 pages
Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85882I (22 February 2013); doi: 10.1117/12.2000583
Show Author Affiliations
Ke Wang, Cornell Univ. (United States)
Tzu-Ming Liu, Wellman Ctr. for Photomedicine, Massachusetts General Hospital (United States)
National Taiwan Univ. (Taiwan)
Juwell Wu, Wellman Ctr. for Photomedicine, Massachusetts General Hospital (United States)
Nicholas G. Horton, Cornell Univ. (United States)
Charles P. Lin, Wellman Ctr. for Photomedicine, Massachusetts General Hospital (United States)
Chris Xu, Cornell Univ. (United States)

Published in SPIE Proceedings Vol. 8588:
Multiphoton Microscopy in the Biomedical Sciences XIII
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)

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