Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser

Evan P. Perillo, Jeremy W. Jarrett, Yen-Liang Liu, Ahmed Hassan, Daniel C. Fernée, John R. Goldak, Andrei Bonteanu, David J. Spence, Hsin-Chih Yeh, Andrew K. Dunn*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)
43 Downloads (Pure)


Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.

Original languageEnglish
Article numbere17095
Pages (from-to)1-8
Number of pages8
JournalLight: Science and Applications
Issue number11
Publication statusPublished - 1 Jun 2017

Bibliographical note

Copyright The Author(s) 2017. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.


  • microscopy
  • nonlinear processes
  • three-dimensional imaging
  • ultrafast lasers


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