Open Access Methodology article

Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

Margaret T Butko1, Mikhail Drobizhev2, Nikolay S Makarov2, Aleksander Rebane2, Brendan C Brinkman1 and Joseph G Gleeson1*

Author Affiliations

1 Departments of Neuroscience and Biomedical Graduate Program, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093 USA

2 Department of Physics, Montana State University, Bozeman, MT 59717 USA

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BMC Biotechnology 2011, 11:20  doi:10.1186/1472-6750-11-20

Published: 2 March 2011



Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.


Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications.


Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM.