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

Abstract

We present a detailed study of the local environmental sensitivity of the commercially available laser dye, Styryl-9M. Positions of the one-photon and two-photon absorption maxima and two-photon absorption minimum of the dye are sensitive to the solvent polarity. In aqueous solution its absorption and fluorescence spectra consist of two peaks whose relative strength depend on the surrounding pH-s. The dye shows one of the highest two-photon absorption cross sections, 700 1300 GM at the peak, among widely available compounds. Comparison of the linear and nonlinear properties shows that its maximum cross section can be described by an effective two-level model. Based on the properties of Styryl-9M we propose a new method of sensing local environment polarity in solutions and biological phantoms. We show that the dye is a promising candidate for two-photon biological imaging and microscopy.

KEYWORDS:

TWO-PHOTON ABSORPTION, STYRYL-9M, PH SENSOR, POLARITY SENSOR, BIOLOGICAL IMAGING

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

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

Two-photon absorption, chloroform

Transition frequency, 10 3 cm -1 30 25 20 15 1000 1x10 5 900 800 1PA absorption Fluorescence 2PA absorption 9x10 4 8x10 4 700 600 500 400 300 200 100 7x10 4 6x10 4 5x10 4 4x10 4 3x10 4 2x10 4 1x10 4 0 300 350 400 450 500 550 600 650 700 750 800 850 900 0 Transition wavelength, nm

1400

Polarity dependence of 2PA

Transition frequency, 10 3 cm -1 32 30 28 26 24 22 20 18 16 14 7 1200 1000 2-Chlorobutane 50% 2-Chlorobutane, 50% Isopropanol Isopropanol Ethanol Acetonitrile 6 5 800 4 600 400 3 2 200 1 0 300 350 400 450 500 550 600 650 Transition wavelength, nm 700 750 800 0

Perrin plot for Styryl-9M

12 10

a

 5 .

1  0 .

5  A 8 6 4 2 0 0.0

2.0x10

-8 4.0x10

-8 6.0x10

-8 8.0x10

-8 1.0x10

-7 1.2x10

-7 1.4x10

-7  /  , s/P

Solvatochromic Stokes shifts for Styryl-9M

7000 6000

F

 1 

i

   

i i

  2

n n

2 2   

i

  2  

i

  5000 4000 3000 0.36

  01  24 .

5  2 .

5 D 0.40

0.44

F(n, )

0.48

0.52

Fluorescence decay kinetics for Styryl-9M

1.0

0.8

Methanol Ethanol Ethylene glycol 0.6

0.4

 Methanol =410 ps  Ethanol =570 ps  Ethylene glycol =470 ps 0.2

0.0

0.0

0.2

0.4

0.6

0.8

Delay, ns 1.0

1.2

1.4

Two-level model description of the two-photon

 2

cross section in chloroform

 2   15 2  4  

f

2 4 01 2

g

  01 01

f

 2   01 2  2 cos 2   1 

g

 3

n

2 3 2

n

2  1  10 3 2  ln   3 10

N hc



A

 01

f

  01

n

2  2 max   01 2  0 .

96  10 20 

hc

  

f S

 

a

3  

S

    01 01

n a

3

f

2 GM  2 max,

calc

 600  180 GM  2 max,

mes

 740  150 GM

pH sensitivity of Styryl-9M: absorption

0.14

0.12

0.10

pH 8.0

pH 8.4

pH 8.8

pH 9.2

pH 9.6

pH 10.0

pH 10.4

0.08

0.06

0.04

0.02

0.00

300 400 500 Wavelength, nm 600 700

pH sensitivity of Styryl-9M: fluorescence

125 100 pH 8.0

pH 8.4

pH 8.8

pH 9.2

pH 9.6

pH 10.0

pH 10.4

75 50 25 0 500 600 700 Wavelength, nm 800 900

5 4 7 6 3 2 1 0 8

pH sensitivity of Styryl-9M

8.0

8.5

9.0

9.5

10.0

8.0

8.5

9.0

pH-level 9.5

10.0

3 2 1 0 10.5

10.5

8 5 4 7 6

Imaging layout

Direction of the laser beam

One-photon sensitivity to local polarity

660 0.34

650 640 2 0.36

630 620 1 610 600 590 580 570 560 0.34

0.36

0.38

3 0.40

0.42

0.44

4 5 0.46

6 7 0.48

8 0.50

660 650 640 630 620 610 600 9 11 590 580 10 570 560 0.48

0.50

0.38

0.40

0.42

0.44

Polarity function 0.46

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

Two-photon sensitivity to local polarity

4 3 6 5 2 1 0 0.34

0.36

0.38

0.40

0.42

F(n,  ) 0.44

0.46

0.48

0.50

2PA sensitivity to local environment in biological phantoms

1.0

0.6

0.5

0.4

0.3

0.9

0.8

0.7

0.2

1 2 Sample number 3 4 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

Conclusions

• Styryl-9M shows one of the strongest two-photon cross section among commercially available chromophores • Despite the lowest energy absorption peak consists of several broadened transitions, it 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 solvent polarity • These properties can be used for two-photon sensing of local environment in biological systems

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Graduating in about 1 year. Looking for a postdoctoral position in fields of nonlinear optics of organic molecules, two-photon biological imaging biophotonics, two-photon photolithography, and photodynamic therapy.

and microscopy,

Important publications

• Makarov N.S., Drobizhev M, Rebane A, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range”,

Opt. Expr.

,

16

storage”, , 2008, 4029-4047.

• Makarov N.S., Rebane A., Drobizhev M., Wolleb H., Spahni H., “Optimizing two-photon absorption for volumetric optical data

J. Opt. Soc. Am. B

,

24

, 2007, 1874-1885.

• Makarov N.S., Bespalov V.G., “Effective method of anti-Stokes generation by quasi-phase-matched stimulated Raman scattering”,

J. Opt. Soc. Am. B

,

22

, 2005, 835-843.

• Drobizhev M., Makarov N.S., Hughes T., Rebane A., “Resonance Enhancement of Two-Photon Absorption in Fluorescent Proteins”, • • • • • • • • • • • • • • •

J. Phys. Chem.

,

111

, 2007, 14051-14054.

• Rebane A., Makarov N.S., Drobizhev M., Spangler B., Tarter E.S., Reeves B.D., Spangler C.W., Meng F., Suo Z., “Quantitative prediction of two photon absorption cross section based on linear spectroscopic properties”,

J. Phys. Chem. C

,

112

, 2008, 7997 8004.

Grants and awards

Soros student 2000, 2001, 2002, 2003 Russian Federation’s President grant 2001 Diploma of Ministry of Education RF for the best scientific student work in natural, technical and humanitarian sciences 2001 RFBR travel grants 2002 Grant of Saint-Petersburg administration for students, aspirants and young specialists-2002 Medal of Russian Academy of Science for the best student work in general physics and astronomy 2002 SPIE Scholarship grant 2003 Diploma for best university graduating student 2003 Dynasty foundation grant 2003 Medal of Ministry of Education RF for the best scientific student work in natural, technical and humanitarian sciences 2003 Grant of Saint-Petersburg administration for students, aspirants and young specialists 2004 Soros aspirant 2004 SPIE Scholarship grant 2006 SPIE BACUS Photomask Scholarship 2007 SPIE D.J. Lovell Scholarship 2008