Local-environment-sensitive two-photon dye

Nikolay S. Makarov, Erich Beuerman, Mikhail Drobizhev, Aleksander Rebane

Department of PhysicsMontana State University, Bozeman, MT

Jean StarkeyDepartment 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 microscopyEndogenous 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

300 350 400 450 500 550 600 650 700 750 800 850 900

0

100

200

300

400

500

600

700

800

900

100030 25 20 15

0

1x104

2x104

3x104

4x104

5x104

6x104

7x104

8x104

9x104

1x105

2, G

M

Transition wavelength, nm

1PA absorption Fluorescence 2PA absorption

Transition frequency, 103 cm-1

, M

-1cm

-1

Polarity dependence of 2PA

300 350 400 450 500 550 600 650 700 750 8000

200

400

600

800

1000

1200

140032 30 28 26 24 22 20 18 16 14

0

1

2

3

4

5

6

7 2,

GM

Transition wavelength, nm

2-Chlorobutane 50% 2-Chlorobutane,

50% Isopropanol Isopropanol Ethanol Acetonitrile

, 1

04 M-1cm

-1

Transition frequency, 103 cm-1

Perrin plot for Styryl-9M

A5.01.5 a

0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 1.0x10-7 1.2x10-7 1.4x10-7

0

2

4

6

8

10

12

0.4

/r-1

/, s/P

0.36 0.40 0.44 0.48 0.52

3000

4000

5000

6000

7000

30%

eth

anol

+ 7

0% w

ater

50%

eth

anol

+ 5

0% w

ater

dich

loro

met

hane

pent

anal

isop

ropa

nol

ethy

lene

gly

col

acet

one

etha

nol

70%

eth

anol

+ 3

0% w

ater

acet

onitr

ile

2-ch

loro

buta

ne

Sto

kes

shift

s, c

m-1

F(n,)

Solvatochromic Stokes shifts for Styryl-9M

22

1,

2

2

i

i

i

i

i n

nnF

D5.25.2401

Fluorescence decay kinetics for Styryl-9M

Methanol=410 ps

Ethanol=570 ps

Ethylene glycol=470 ps

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.40.0

0.2

0.4

0.6

0.8

1.0

Flu

ore

sce

nce

, a.u

.

Delay, ns

Methanol Ethanol Ethylene glycol

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

GM1096.0

01

230120max

2 n

faS

12

32

2

n

nf

GM180600max,2 calc

GM150740max,2 mes

21cos215

22 22

01

2

012

44

2 gnch

f

2

013

013

01

2

012

10ln103

fN

nhcg

A

Dfahc S

3

2

01

pH sensitivity of Styryl-9M: absorption

300 400 500 600 7000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Ab

sorp

tion

, a.u

.

Wavelength, nm

pH 8.0 pH 8.4 pH 8.8 pH 9.2 pH 9.6 pH 10.0 pH 10.4

pH sensitivity of Styryl-9M: fluorescence

500 600 700 800 9000

25

50

75

100

125F

luo

resc

en

ce, a

.u.

Wavelength, nm

pH 8.0 pH 8.4 pH 8.8 pH 9.2 pH 9.6 pH 10.0 pH 10.4

pH sensitivity of Styryl-9M

8.0 8.5 9.0 9.5 10.0 10.50

1

2

3

4

5

6

7

88.0 8.5 9.0 9.5 10.0 10.5

0

1

2

3

4

5

6

7

8F

luo

resc

en

ce in

ten

sity

(7

95

nm

) /

Flu

ore

sce

nce

inte

nsi

ty (

65

0 n

m)

pH-level

Imaging layoutDirection of the laser beam

One-photon sensitivity to local polarity

0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50

560

570

580

590

600

610

620

630

640

650

660

0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50

560

570

580

590

600

610

620

630

640

650

660

11

10

98

7

6

5

4

3

2

1

Cen

tral

abs

orpt

ion

wav

elen

gth,

nm

Polarity function

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 waterThe 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

0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.500

1

2

3

4

5

6

F(9

00

)/F

(10

00

)

F(n, )

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 overlay2 – 25 l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen3 – 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 mixture4 – 25 l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen, 1 l liposin

1 2 3 40.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

F(9

50

)/F

(11

00

)

Sample number

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