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Local-environment-sensitive two-photon dye. Nikolay S. Makarov , Erich Beuerman, Mikhail Drobizhev, Aleksander Rebane Department of Physics Montana State University, Bozeman, MT Jean Starkey Department of Microbiology Montana State University, Bozeman, MT. Outline.
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Local-environment-sensitive two-photon dye Nikolay S. Makarov, Erich Beuerman, Mikhail Drobizhev, Aleksander Rebane Department of Physics Montana State University, Bozeman, MT Jean Starkey Department of Microbiology Montana State University, Bozeman, MT
Outline • Advantages of two-photon microscopy • Properties of molecular probes for two-photon microscopy • Two-photon absorption of Styryl-9M • Linear photophysical properties of Styryl-9M • pH sensitivity of Styryl-9M • One-photon sensitivity to local polarity • Two-photon sensitivity to local polarity • Two-photon sensitivity to local environment in biological phantoms
Advantages of two-photon microscopy • Inherent 3D resolution • Larger penetration depth • Lower scattering • Lower photodamage • Lower absorption by intrinsic molecules • Lower autofluorescence background Probes for two-photon microscopy
Fluorescence decay kinetics for Styryl-9M Methanol=410 ps Ethanol=570 ps Ethylene glycol=470 ps
Two-level model description of the two-photon cross section in chloroform
Imaging layout Direction of the laser beam
One-photon sensitivity to local polarity 1 – 2-chlorobutane; 2 – dichloromethane; 3 – pentanal; 4 – isopropanol; 5 – ethylene glycol; 6 – acetone; 7 – ethanol; 8 – mixture of 70% ethanol + 30% DI water; 9 – mixture of 50% ethanol + 50% DI water; 10 – acetonitrile; 11 – mixture of 30% ethanol + 70% DI water Two “unknown” samples: (1) mixture of 50% 2-chlorobutane + 50% isopropanol (2) mixture of 40% ethanol + 60% DI water The determined polarity function is 0.386 for (1) and 0.497 for (2) which is less than 2% off the “true” values
2PA sensitivity to local environment in biological phantoms 1 – 10 l of 5mg/ml Styryl-9M dissolved in DMSO, 5105 Mouse embryo fibroblast cells, 0.6 ml setting solution, 3 ml rat tail collagen, 1 l linoleic/oleic acid mixture, 1 ml serum and phenol red –free medium overlay 2 – 25 l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen 3 – 25 l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen, 1 l linoleic/oleic acid mixture 4 – 25 l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen, 1 l liposin
Conclusions • Styryl-9M shows one of the strongest two-photon cross section among commercially available chromophores • The lowest energy absorption peak can be considered as an effective two-level system • Both absorption and fluorescence spectra of the dye are highly sensitive to pH • Both 1PA and 2PA peaks of the dye are sensitive to the polarity • These properties can be used for two-photon sensing of local environment in biological systems • Might expect to be able to differentiate malignant from normal cells using polarity images with Styryl-9M -like compounds