Localenvironmentsensitive twophoton dye

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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
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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

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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
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Two-photon absorption, chloroform
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Polarity dependence of 2PA
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Perrin plot for Styryl-9M
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Solvatochromic Stokes shifts for Styryl-9M
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Fluorescence decay kinetics for Styryl-9M
?Methanol410 ps ?Ethanol570 ps ?Ethylene
glycol470 ps
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Two-level model description of the two-photon
cross section in chloroform
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pH sensitivity of Styryl-9M absorption
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pH sensitivity of Styryl-9M fluorescence
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pH sensitivity of Styryl-9M
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Imaging layout
Direction of the laser beam
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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
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Two-photon sensitivity to local polarity
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2PA sensitivity to local environment in
biological phantoms
1 10 ?l of 5mg/ml Styryl-9M dissolved in DMSO,
5?105 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
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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