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Abstract
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.
Highlights
Fluorescent proteins (FP) are widely used as genetically encoded fluorescent reporters in biomedical research [1]
Using a broadband femtosecond laser and ultrafast phase-shaping techniques, we have developed a two-photon imaging modality allowing selective excitation of three FPs with distinct fluorescence spectra
By combining this with linear unmixing from two PMT channels, the fluorescence signal of each of the three FPs was restored with high separation
Summary
Fluorescent proteins (FP) are widely used as genetically encoded fluorescent reporters in biomedical research [1]. While the use of transform-limited pulses enables simultaneous excitation of multiple FPs, it does not provide selectivity and excites them at fixed efficiency ratios This is undesirable as adjusting relative FP brightness becomes impossible, and undesirable signals such as autofluorescence can be enhanced. We report using phase-shaping of ultrafast pulses to selectively excite three FPs expressed in mammalian cells By combining this with post-imaging linear unmixing, we demonstrate the feasibility of using a single broadband laser and SLM pulse-shaper to achieve three-color 2PFM in live cells. The relatively less expensive price, ease of alignment, and flexibility in choosing FPs make this two-photon modality attractive for use in many biomedical laboratories This method could be readily extended beyond three-color imaging with even broader bandwidth commercially available Titanium:Sapphire oscillators
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