Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser.
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.
Applied Physics Program, University of Michigan 450 Church St, Ann Arbor MI 48109, USA.
Meredith H
MH
Brenner
Dawen
D
Cai
Joel A
JA
Swanson
Jennifer P
JP
Ogilvie
D000818:Animals; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation*; D007834:Lasers; D008164:Luminescent Proteins / Q000032:analysis* / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D008164:Luminescent Proteins