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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Equipment Design
  • Equipment Failure Analysis
  • Humans
  • Image Enhancement / instrumentation*
  • Lasers*
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence, Multiphoton / instrumentation*
  • Microscopy, Fluorescence, Multiphoton / methods
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Luminescent Proteins