PMID-21527931 Two-photon absorption properties of fluorescent proteins
- Significant 2-photon cross section of red fluorescent proteins (same chromophore as DsRed) in the 700 - 770nm range, accessible to Ti:sapphire lasers ...
- This corresponds to a transition
- But but, photobleaching is an order of magnitude slower when excited by the direct transition (but the fluorophores can be significantly less bright in this regime).
- Quote: the photobleaching of DsRed slows down by an order of magnitude when the excitation wavelength is shifted to the red, from 750 to 950 nm (32).
- See also PMID-18027924
- Further work by same authors: Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins
- " TagRFP possesses the highest two-photon cross section, σ2 = 315 GM, and brightness, σ2φ = 130 GM, where φ is the fluorescence quantum yield. At longer wavelengths, 1000–1100 nm, tdTomato has the largest values, σ2 = 216 GM and σ2φ = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3–4 times improvement in two-photon brightness."
- "Single-photon properties of the FPs are poor predictors of which fluorescent proteins will be optimal in two-photon applications. It follows that additional mutagenesis efforts to improve two-photon cross section will benefit the field."
- 2P cross-section in both the 700-800nm and 1000-1100 nm range corresponds to the chromophore polarizability, and is not related to 1p cross section.
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- This can be useflu for multicolor imaging: excitation of the higher S0 → Sn transition of TagRFP simultaneously with the first, S0 → S1, transition of mKalama1 makes dual-color two-photon imaging possible with a single excitation laser wavelength (13)
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- Why are red GECIs based on mApple (rGECO1) or mRuby (RCaMP)? dsRed2 or TagRFP are much better .. but maybe they don't have CP variants.
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- from https://elifesciences.org/articles/12727
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