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. 2017 Jun 19;7:3792. doi: 10.1038/s41598-017-03359-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Multicolor two-photon efficient imaging of multiple endogenous fluorophores during early stages of zebrafish embryo development (a) Energy diagrams of two-photon excited fluorescence (2PEF) for blue and red fluorophores, two-color two-photon excited fluorescence (2c-2PEF) for green fluorophores, second harmonic generation (SHG), sum frequency generation (SFG) and four wave mixing (FWM). (b) Bandpass filters are chosen to select the emission of blue, green and red fluorophores rejecting coherent signals such as SHG, SFG and FWM. (c) Images of the blue, green, red fluorescence channel and SHG of the same zebrafish embryo at three different time points of development. Images of merged channels represent a shift in the spectroscopic characteristics of the yolk. (d) Multicolor two-photon and SHG images of the zebrafish embryo at 4 cell stage. Time per pixel, 40 µs. This experiment has been repeated in three independent samples.