Multi-color femtosecond source for simultaneous excitation of multiple fluorescent proteins in two-photon fluorescence microscopy

Ke Wang; Tzu-Ming Liu; Juwell Wu; Nicholas G. Horton; Charles P. Lin; Chris Xu

doi:10.1117/12.2000583

22 February 2013 Multi-color femtosecond source for simultaneous excitation of multiple fluorescent proteins in two-photon fluorescence microscopy

Ke Wang, Tzu-Ming Liu, Juwell Wu, Nicholas G. Horton, Charles P. Lin, Chris Xu

Author Affiliations +

Proceedings Volume 8588, Multiphoton Microscopy in the Biomedical Sciences XIII; 85882I (2013) https://doi.org/10.1117/12.2000583
Event: SPIE BiOS, 2013, San Francisco, California, United States

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.

Citation Download Citation

Ke Wang, Tzu-Ming Liu, Juwell Wu, Nicholas G. Horton, Charles P. Lin, and Chris Xu "Multi-color femtosecond source for simultaneous excitation of multiple fluorescent proteins in two-photon fluorescence microscopy", Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85882I (22 February 2013); https://doi.org/10.1117/12.2000583

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available