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Opt Express. 2013 Jul 15;21(14):17256-64. doi: 10.1364/OE.21.017256.

Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser.

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  • 1Applied Physics Program, University of Michigan 450 Church St, Ann Arbor MI 48109, USA.


Imaging multiple fluorescent proteins (FPs) by two-photon microscopy has numerous applications for studying biological processes in thick and live samples. Here we demonstrate a setup utilizing a single broadband laser and a phase-only pulse-shaper to achieve imaging of three FPs (mAmetrine, TagRFPt, and mKate2) in live mammalian cells. Phase-shaping to achieve selective excitation of the FPs in combination with post-imaging linear unmixing enables clean separation of the fluorescence signal of each FP. This setup also benefits from low overall cost and simple optical alignment, enabling easy adaptation in a regular biomedical research laboratory.

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