1. 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

2. 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

3. 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 Endogenous fluorophores (NADH, NADPH, retinol, lipofucsin, etc.) + natural source of 2PA- excited fluorescence + fills the whole visible spectrum - low 2PA cross sections Fluorescent proteins + can be genetically encoded + high flexibility studying small animals - small loading concentration - substantial size - faster photobleaching - higher cost Exogenous fluorophores + high 2PA cross sections + possible targeting + high stability + small size + possible near-infrared fluorescence - delivery problems - non-specific dying

4. Two-photon absorption, chloroform

5. Polarity dependence of 2PA

6. Perrin plot for Styryl-9M

7. Solvatochromic Stokes shifts for Styryl-9M

8. Fluorescence decay kinetics for Styryl-9M  Methanol =410 ps  Ethanol =570 ps  Ethylene glycol =470 ps

9. Two-level model description of the two-photon cross section in chloroform

10. pH sensitivity of Styryl-9M: absorption

11. pH sensitivity of Styryl-9M: fluorescence

12. pH sensitivity of Styryl-9M

13. Imaging layout Direction of the laser beam

14. 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

15. Two-photon sensitivity to local polarity

16. 2PA sensitivity to local environment in biological phantoms 1 – 10  l of 5mg/ml Styryl-9M dissolved in DMSO, 5  10 5 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

17. 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