Symmetrical dyes with large two-photon absorption cross-sections

A two-photon absorbing chromophore of the formula EQU E--Ar--E wherein the Ar core is selected from the group consisting of ##STR1## wherein R.sub.1 and R.sub.2 are alkyl groups having 8 to 12 carbon atoms, and wherein R.sub.1 and R.sub.2 are the same or different, and wherein E is a moiety selected from the group consisting of ##STR2##

RIGHTS OF THE GOVERNMENT 
The invention described herein may be manufactured and used by or for the 
Government of the United States for all governmental purposes without the 
payment of any royalty. 
BACKGROUND OF THE INVENTION 
In ordinary fluorescence microscopy, defocused images outside the depth of 
focus are superimposed on an image formed on the focal plane. This 
globally lowers the contrast of microscopic image, which makes 
determination of fluorescence intensity difficult. 
Confocal microscopy offers several advantages over conventional microscopy. 
The shallow depth of field, generally about 0.5 to 1.5 .mu.m, of confocal 
microscopes allows information to be collected from a well defined optical 
section rather than from most of the specimen as in conventional light 
microscopy. Consequently, out-of-focus fluorescence is virtually 
eliminated, which results in an increase in contrast, clarity and 
detection. 
In a point scanning confocal system, the microscope lens focus the laser 
light on one point in the specimen at a time, i.e., the focal point. The 
laser moves rapidly from point to point to produce a scanned image. Very 
little of the laser light falls on other points in the focal plane. Both 
fluorescent and reflected light from the sample pass back through the 
microscope. The microscope and the optics of the scanner compartment focus 
the fluorescent light emitted from the focal point to a secont point, 
called the confocal point. A pinhole aperature, located at the confocal 
point, allows light from the focal point to pass through to a detector. 
Light emitted from outside the focal point is rejected by the aperature. 
Accordingly, only the image near the focal plane inside the sample is 
obtained as a microscopic image. 
In two-photon absorption excitation type laser scanning fluorescence 
microscopy, a laser beam forms an optical spot having a high energy 
density and the optical spot three-dimensionally scans the inside of a 
sample in the same manner as in confocal laser scanning fluorescence 
microscopy. Because of the arrangement, fluorescence due to excitation 
based on two-photon absorption appears only from a point where the optical 
spot is located inside the sample but no fluorescence due to excitation 
based on two-photon absorption appears from other portions. Therefore, 
there appears no defocused image other than one on the focal plane, which 
improves the contrast of the microscopic image. 
Two-photon excitation is made possible by the combination of (a) the very 
high, local, instantaneous intensity provided by the tight focusing 
available in a laser scanning microscope, wherein the laser can be focused 
to diffraction-limited waist of less than 1 micron in diameter, and (b) 
the temporal concentration of a pulsed laser. A high intensity, long 
wavelength, monochromatic light source which is focusable to the 
diffraction limit such as a colliding-pulse, mode-locked dye laser, 
produces a stream of pulses, with each pulse having a duration of about 
100 femtoseconds (100.times.10.sup.-15 seconds) at a repetition rate of 
about 80 MHz. These subpicosecond pulses are supplied to the microscope, 
for example by way of a dichroic mirror, and are directed through the 
microscope optics to a specimen, or target material, located at the object 
plane of the microscope. Because of the high instantaneous power provided 
by the very short duration intense pulses focused to the diffraction 
limit, there is an appreciable probability that a fluorophore (a 
fluorescent dye), contained in the target material, and normally excitable 
by a single high energy photon having a short wavelength, typically 
ultraviolet, will absorb two long wavelength photons from the laser source 
simultaneously. This absorption combines the energy of the two photons in 
the fluorophore molecule, thereby raising the fluorophore to its excited 
state. When the fluorophore returns to its normal state, it emits light, 
and this light then passes back through the microscope optics to a 
suitable detector. 
The probability of absorption of two long wavelength photons from the laser 
source simultaneously is dependent upon the two-photon cross-section of 
the dye molecule. 
Accordingly, it is an object of the present invention to provide 
chromophores having large two-photon cross-sections. 
Other objects and advantages of the present invention will be apparent to 
those skilled in the art. 
SUMMARY OF THE INVENTION 
In accordance with the present invention there are provided symmetrical 
chromophores of the formula: 
EQU E--Ar--E 
wherein the Ar core is selected from the group consisting of 
##STR3## 
wherein R.sub.1 and R.sub.2 are alkyl groups having 8 to 12 carbon atoms, 
and wherein R.sub.1 and R.sub.2 may be the same or different, and wherein 
E is a moiety selected from the group consisting of 
##STR4##

The following examples illustrate the invention: 
EXAMPLE I 
Preparation of 2,7-dibromo-9,9-di-n-decyl-9H-fluorene 
Fluorene (27.70 g, 0.016 mol) and hexane (620 ml) were added to a 
three-necked round bottom flask equipped with a reflux condenser, a 
mechanical stirrer, and an addition funnel containing n-butyllithium (100 
ml of a 2.0M solution in cyclohexane, 0.2 mol). The fluorene was dissolved 
in hexane by stirring and warming the solution with a heat gun. When all 
the fluorene was in solution, the solution was allowed to cool to room 
temperature. The butyllithium solution was then added dropwise over a 45 
min period at room temperature. The rust-colored solution was stirred an 
additional hour before adding N, N, N', N',-tetramethyleneethylenediamine 
(TMEDA) (23.20 g, 0.2 mol) dropwise through a clean addition funnel. The 
solution was stirred an additional hour before 1-bromodecane (44.24 g, 0.2 
mol) was added through another clean addition funnel. The pale orange 
solution was refluxed for 4 hours and then cooled to room temperature 
using an ice bath. A second equivalent of butyllithium (100.0 ml of a 2.0M 
solution in cyclohexane, 0.2 mol) was added at room temperature and was 
allowed to stir for an additional hour. Finally, 1-bromodecane (44.24 g, 
0.2 mol) was added dropwise and the solution heated at reflux for an 
additional 6 hours. 
The solution was cooled to room temperature and the resulting solid was 
vacuum filtered and the crystals rinsed with hexane. The solvent was 
removed under reduced pressure to yield a yellow oil that was purified by 
column chromatography on silica gel using hexane as the eluent. The 
product was futher purified by vacuum distillation (bp 257.degree. C. (2 
Torr)) to remove any excess bromodecane to afford 
9,9-di-n-decyl-9H-fluorene as clear yellow oil in 93% yield. Mass Spec. 
m/z 446 (M.sup.+), 418 (M--C.sub.2 H.sub.4), 305 (M--C.sub.10 H.sub.21). 
Elemental Analysis: Calculated for C.sub.33 H.sub.50 : C, 88.72; H, 11.28. 
Found: C, 88.47; H, 10.93. 
A solution of 9,9-di-n-decyl-9H-fluorene (21.33 g, 0.047 mol), iodine (0.12 
g, 0.47 mmol), and methylene chloride (170 ml) was stirred magnetically in 
a single-necked round bottom flask covered with aluminum foil to exclude 
light. Elemental bromine (5.18 ml, 0.10 mol) in methylene chloride (20 ml) 
was pipetted into an addition funnel which was then added dropwise to the 
reaction mixture over a period of 15 minutes. Residual bromine solution 
was rinsed out of the addition funnel into the reaction mixture using 20 
ml of additional methylene chloride and the reaction was allowed to stir 
for 20 hours at room temperature. An aqueous solution (15% by weight) of 
sodium bisulfite (NaHSO.sub.3) was then added to the reaction mixture and 
the resulting two phases allowed to stir for 30 min. The organic layer was 
then separated and washed 3 times with an equal volume of water and then 
dried over anhydrous magnesium sulfate. The solution was filtered, 
concentrated and the resulting solid recrystallized from absolute ethanol 
to yield 2,7-dibromo-9,9-di-n-decyl-9H-fluorene as to white crystals, mp 
38.1.degree.-38.4.degree. C. in 82.8% yield. Mass Spec. m/z 602, 604, 606 
(M.sup.+), 461, 463, 465 (M--C.sub.10 H.sub.21), 382, 384 (461-Br). 
Elemental Analysis: Calculated for C.sub.33 H.sub.48 Br.sub.2 : C, 65.56; 
H, 8.00; Br, 26.44. Found: C, 64.99; H, 8.21; Br, 27.25. 
EXAMPLE II 
Preparation of 2,7-bis-(2-thienyl)-9,9-di-(n-decyl)-9H-fluorene 
2-(tributylstannyl)thiophene (5.80 ml, 0.018 mol) was added under nitrogen 
directly to a single-necked round bottom flask equipped with a condenser 
and a magnetic stir bar that had been previously dried in an oven at 
110.degree. C. The 2,7-dibromo-9,9-di-n-decyl-9H-fluorene (5.26 g, 8.70 
mmol) was weighed into a beaker and dissolved in toluene (14 ml) which had 
been freshly distilled and degassed with nitrogen. The resulting fluorene 
solution was pipetted into the round bottom flask and the beaker rinsed 
twice with 3 ml portions of degassed toluene. Pd(PPh.sub.3).sub.4 (0.50 g, 
0.43 mmol) and PdCl.sub.2 (PPh.sub.3).sub.2 (0.31 g, 0.44 mmol) were added 
to the reaction flask and the solution refluxed until it turned black. The 
toluene was removed under reduced pressure and the residue dissolved in 
hexane. The hexane solution was stirred vigorously with a 100 ml of a 2% 
aqueous KF solution for 3 hours. The organic layer was separated, washed 
twice with an equal volume of water, dried over anhydrous MgSO.sub.4, 
filtered and concentrated. The crude product was purified by column 
chromotography on silica gel using hexane as the eluent. The purified waxy 
solid compound (mp 59.9.degree.-60.8.degree. C.) was isolated in a 70% 
yield. Mass Spec. m/z 610 (M.sup.+), 469 (M--C.sub.10 H.sub.21), Elemental 
Analysis: Calculated for C.sub.41 H.sub.52 S.sub.2 : C, 80.60, H, 8.91, S, 
10.49. Found: C, 80.55, H, 8.97, S, 10.08. 
EXAMPLE III 
3,4-Bis(decyloxy)-5-(benzothiazol-2-yl)thiophene-2-carboxylic acid 
A mixture of the 3,4-bis(decyloxy)-2,5-thiophene dicarboxyllic acid (23.5 
g), dichlorobenzene (130 ml), 2-aminothiophenol (8.9 g), 
trimethylsilylpolyphosphate (60 g), and tri-n-butylamine was heated at 
100.degree. C. for 22 hr under nitrogen. After cooling, the reaction 
mixture was diluted with water, the chlorobenzene layer separated, dried 
over magnesium sulfate and concentrated. The residual waxy solid was 
chromatographed over 150 g of silica gel. Elution with heptane gave the 
bis-benzothiazole by-product. 12.74 g (40% yield), mp 
76.degree.-77.8.degree. C. Futher elution with toluene-heptane 
mixtures(1:3 and 1:2) gave the desired monocarboxyllic acid which was 
recrystallized from toluene-heptane to give 11.27 g, (40.5% yield), mp 
139.7.degree.-141.7.degree. C. 
EXAMPLE IV 
2-Bromo-3,4-bis(decyloxy)-5-(benzothiazol-2-yl)-thiophene 
To a magnetically stirred mixture of the monocarboxyllic acid (17.2 g), 
potassium carbonate (8.4 g), and water (300 ml) held at 45.degree. C., 
bromine (8.4 g) was added over 15 min. After 45 min at 50.degree. C. the 
mixture was cooled to room temperature and treated with 10% sodium 
hydroxide, water, and saturated sodium chloride solution. After drying, 
the toluene solution was concentrated and the residual oil was 
chromatographed over 200 g of silica gel. Elution with 1:3 toluene-heptane 
gave an oily product which solidified upon standing with methanol to give 
16.85 g (91% yield), to mp 42.degree.-43.degree. C. Mass Spec: m/z 480-482 
(M.sup.+ --C.sub.9 H.sub.19). Elemental Analysis: Calculated for C.sub.31 
H.sub.46 BrNO.sub.2 S.sub.2 : C, 61.16, H, 7.62, Br, 13.14, N, 2.30, and 
S, 10.51%. Found: C, 60.85, H, 7.70, Br, 13.48, N, 2.29, and S, 10.42 %. 
EXAMPLE V 
3,4,3',4'-tetradecyloxy-5,5'-bis(benzothiazol-2-yl)-2,2'-bithiophene 
A solution of 2-bromo-3,4-bis(decyloxy)-5-(benzothiazol-2-yl)-thiophene 
(12.2 g 20 mmol), and hexabutyl ditin (5.8 g, 10 mmol) in dioxane (100 
ml), (freshly distilled from sodium after treatment with potassium 
hydroxide), was degassed by bubbling nitrogen through the solution for 20 
min. Under nitrogen, tetrakistriphenylphosphinopalladium (0.468 g) was 
added and the mixture was heated at 100.degree. C. for 20 hr. After 
cooling, pyridine (5 ml) was added and the resulting mixture allowed to 
stand for 1 hr. The separated solids were filtered and the filtrate 
diluted with an equal volume of water. The mixture was extracted with an 
equal volume of toluene, dried over magnesium sulfate and concentrated. 
The residue was combined with the initial solids and chromatographed over 
250 g of silica gel. Elution with 3:1 and 2:1 heptane:toluene gave the 
desired product which was recrystallized from heptane to give 7.02 g 
(66.5% yield) mp 93.9.degree.-95.degree. C. Mass Spec. m/z 916 (M.sup.+ 
--C.sub.10 H.sub.20). Elemental Analysis: Calculated for C.sub.62 H.sub.92 
N.sub.2 O.sub.4 S.sub.4 : C, 70.36, H, 8.77, N, 2.65, and S, 12.10%. 
Found: C, 69.98, H, 8.90, N, 2.59, and S, 11.98 %. 
EXAMPLE VI 
2,7-bis(4-pyridyl)-9,9-di-n-decyl-9H-fluorene 
4-(tri-n-butylstannyl)pyridine (8.76 g, 23.8 mmol) was added directly to a 
single-necked round bottom flask equipped with a condenser and a magnetic 
stir bar that had been dried in an oven at 110.degree. C. overnight. 
2,7-dibromo-9,9-di-n-decyl-9H-fluorene (6.85 g, 11.33 mmol) was weighed 
into a beaker and dissolved in dry toluene (30 ml) degassed with nitrogen. 
The fluorene solution was pipetted into the round bottom flask and the 
beaker rinsed with 25 ml portions of degassed toluene. Pd(PPh.sub.3).sub.4 
(0.28 g, 0.24 mmol), PdCl.sub.2 (PPh.sub.3).sub.4 (1.67 g, 2.38 mmol) and 
triphenylphosphine (1.37 g, 5.22 mmol) were added to the reaction flask 
and the solution heated at reflux until it turned black. The reaction 
mixture was cooled to room temperature, the toluene removed under reduced 
pressure and the residue dissolved in hexane. The hexane solution was 
stirred vigorously with a 100 ml of a 2% aqueous KF solution for 3 hours. 
The organic layer was separated, washed twice with an equal volume of 
water, dried over anhydrous MgSO.sub.4, filtered and concentrated. The 
crude product was purified by column chromatography on silica gel using 
30/70 THF/hexane. The purified compound was isolated in 18.7% yield as 
light brown oil. Mass Spec. m/z 600 (M.sup.+), 459 (M--C.sub.10 H.sub.21), 
167 (C.sub.11 H.sub.7 N.sub.2)+. Elemental Analysis: Calculated for 
C.sub.43 H.sub.56 N.sub.2 : C, 85.95, H, 9.39, N, 4.66. Found: C, 85.59, 
H, 8.97, N, 4.16. 
EXAMPLE VII 
4,4'-(9,9-bis-n-decyl-2,7-fluorenediyldi-2,1 -ethenediyl)bis-pyridine 
2,7-Dibromo-9,9-di-n-decyl-9H-fluorene (10.0 g, 16.5 mmol), 
tri-o-tolylphosphine (2.01 g, 6.6 mmol), degassed triethylamine (40 ml) 
and 4-vinylpyridine (5.35 ml, 49.62 mmol) was added to a single-necked 
round bottom flask under to nitrogen equipped with a magnetic stir bar and 
a reflux condenser. The palladium catalyst (0.19 g, 0.83 mmol) was added 
and the solution heated at 95.degree. C. for 18 hours. The reaction 
mixture was cooled to room temperature and the solvent removed under 
reduced pressure. The residue was dissolved in methylene chloride, and the 
organic layer washed three times with equal volumes of water, dried over 
anhydrous MgSO.sub.4, filtered and concentrated. The product was purified 
by column chromotography on alumina using 30/70 ethyl acetate/hexane to 
yield the product as a yellow oil in 71.48% yield.Mass Spec. m/z 652 
(M.sup.+), 511 (M--C.sub.10 H.sub.21). Elemental Analysis: Calculated for 
C.sub.47 H.sub.60 N.sub.2 : C, 86.45, H, 9.26, N, 4.29. Found: C, 86.50, 
H, 8.84, N, 3.78. 
EXAMPLE VIII 
Determination of 2-Photon Absorption Cross-Section 
The two photon absorption coefficients .beta. and the molecular two-photon 
cross-section .sigma..sub.2 were determined from an experimental 
measurement of the transmitted intensity of a laser beam at 798 nm as a 
function of the incident intensity. According to the basic theoretical 
consideration, the two-photon absorption (TPA) induced decrease in 
transmissivity can be expressed as 
EQU I(L)=I.sub.O /(1+I.sub.O L.beta.) (1) 
where I(L) is the transmitted beam intensity, I.sub.O the incident beam 
intensity, L the thickness of the sample, and .beta. is the TPA 
coefficient of the sample medium. In the to derivation of the above 
equation it is assumed that the linear attenuation of the medium can be 
neglected and the beam has a nearly uniform transverse intensity 
distribution within the medium. The TPA coefficient can be determined by 
measuring the transmitted intensity I(L) as a function of various incident 
intensities I.sub.O for a given medium with a given L value. The TPA 
coefficient .beta. (in units of cm/GW) of a given sample is determined by 
EQU .beta.=.sigma..sub.2 N.sub.O =.sigma..sub.2 N.sub.A d.sub.0 
.times.10.sup.-3(2) 
where N.sub.O is the molecular density of the material being measured (in 
units of 1/cm.sup.3), (.sigma..sub.2 the molecular TPA cross-section of 
the same material (in units of cm.sup.4 /GW), d.sub.0 is the concentration 
of the material (in units of M/L) and finally N.sub.A, Avogadro's number. 
For known .beta. and d.sub.0, the value of .sigma..sub.2 can be easily 
calculated from equation (2). 
In practice, various optical detectors such as photodiodes, 
photomultipliers, photometers and optical power meters can be used to 
measure the incident beam intensity I.sub.O and the transmitted beam 
intensity I(L) separately. The change of the I.sub.O values can be done by 
using variable optical attenuators (such as neutral filters or rotatable 
polarizing prisms), or by varying the beam cross-section of the input 
laser beam (by changing the relative distance of the sample to the focal 
point of the input beam). The input laser beam used for the experimental 
measurements was a pulsed laser dye system with a wavelength of 800 nm, a 
spectral width of 1-10 nm, a pulse duration of 8-10 ns, a beam size 
(before focusing lens) of 3-5 nm, a beam divergency of 1.2-1.5 mrad, and a 
repetition rate between 1-30 Hz. 
______________________________________ 
Experimentally Determined TPA coefficients (.beta.) and cross-sections 
(.sigma.) 
Compound 
Concentration 
.beta. .sigma..sub.2 
of Example 
M/L cm/GW (.times.10.sup.-20) cm.sup.4 /GW) 
Solvent 
______________________________________ 
II 0.0418 0.058 0.230 THF 
V 0.0567 5.000 14.600 THF 
VI 0.0935 0.085 0.152 THF 
VII 0.0935 0.491 0.873 THF 
______________________________________ 
These data clearly show increased size of the 2-photon cross-section as 
compared to most state-of-the-art organic compounds. The increased 
cross-section, when coupled with strong upconverted fluorescence, makes 
these chromophores more useful in fluorescent imaging application such as 
2-photon laser scanning confocal microscopy where strong fluorescence is 
needed to obtain high resolution. Large 2-photon absorption cross-sections 
also lead to greatly improved optical limiting behavior. In addition the 
long chain alkyl groups incorporated into these materials lead to very 
high solubility in organic solvents and good compatibility with organic 
polymers. Solubility of these chromophores is generally at least 0.01 and 
up to about 35% in common organic solvents. 
Two-photon absorbing dyes which exhibit upconverted fluorescence have also 
found use in other areas of photonic technology. These include 2-photon 
pumped upconverted lasing, 2-photon confocal microscopy, 2-photon 
photodynamic therapy, 2-photon optical power limiting, and 3D optical data 
storage. 
Various modifications may be made in the instant invention without 
departing from the spirit and scope of the appended claims.