Two-photon photosensitized production of singlet oxygen

Article abstract of DOI:10.1021/ja003468a

Singlet molecular oxygen (a¹δ_g) has been produced and optically detected upon two-photon nonlinear excitation of a sensitizer with a focused laser beam. The experiments were performed using toluene solutions with either a substituted

Full text of DOI:10.1021/ja003468a

J. Am. Chem. Soc. 2001, 123, 1215-1221
1215
Two-Photon Photosensitized Production of Singlet Oxygen
Peter K. Frederiksen,† Mikkel Jørgensen,‡ and Peter R. Ogilby*,†
Contribution from the Department of Chemistry, Aarhus UniVersity, DK-8000 Aarhus, Denmark,
and Condensed Matter Physics and Chemistry Department, Risø National Laboratory,
DK-4000 Roskilde, Denmark
ReceiVed September 22, 2000. ReVised Manuscript ReceiVed NoVember 20, 2000
Abstract: Singlet molecular oxygen (a1∆g) has been produced and optically detected upon two-photon nonlinear
excitation of a sensitizer with a focused laser beam. The experiments were performed using toluene solutions
with either a substituted difuranonaphthalene or a substituted distyryl benzene as the sensitizer. The data indicate
that the two-photon absorption cross sections of the difuranonaphthalenes are comparatively large and depend
significantly on the functional groups attached to the chromophore. The time-resolved 1270 nm phosphorescence
signals used to characterize the production of singlet oxygen are limited in much the same way as signals
from other two-photon spectroscopic studies (e.g., weak signals that can be masked by scattered radiation).
Nevertheless, the two-photon singlet oxygen signals also reflect the unique advantages of this nonlinear optical
technique (e.g., depth penetration in the sample afforded by irradiation in a spectral region void of the more
dominant one-photon linear transitions and spatial resolution afforded by irradiation with a focused laser beam).
Introduction
This can be important in photodynamic therapy, for example.6
Although the use of two-photon nonlinear optical techniques
to populate excited electronic states is well established, it has
heretofore not been demonstrated as a viable pathway for the
photosensitized production of singlet oxygen.
The photosensitized production of singlet molecular oxygen
(a1∆g) has important ramifications in disciplines that range from
photobiology to polymer science.1 In the vast literature on singlet
oxygen photosensitization, one finds that sensitizer excitation
is invariably achieved via a one-photon linear transition between
the ground state, S0, and a singlet excited state, Sn. Rapid
relaxation of the Sn state initially populated yields the lowest
excited singlet state of the sensitizer, S1. Although ground-state
oxygen can quench S1 of some molecules, singlet oxygen is
generally more efficiently generated upon oxygen quenching
of the sensitizer triplet state, T1, produced upon S1 f T1
intersystem crossing.
Although the mechanism and selection rules of excited-state
population in a two-photon pumping scheme differ from those
in a one-photon pumping scheme, and although the initial states
populated as a result of these transitions are likewise different,
both processes ultimately result in the creation of the lowest
excited singlet state, S1, via internal conversion. A key difference
between these respective processes that, in turn, reflects the
respective mechanisms of excitation will be the yield with which
this lowest singlet state is produced. In general, the S1 yield is
∼7-8 orders of magnitude lower for the two-photon process
than for the one-photon process.7 Nevertheless, once this singlet
state is formed, subsequent events such as fluorescence and/or
intersystem crossing will be independent of whether a one- or
two-photon pumping scheme was used.6 Thus, if one is able to
(1) overcome the limitations associated with low excited-state
yields and the concomitant weak spectroscopic signals and (2)
identify a molecule that not only can efficiently produce singlet
oxygen but also has an appreciable two-photon absorption cross
section, one should, in principle, be able to demonstrate that
singlet oxygen can also be formed as a result of a two-photon
photosensitized process. We now report that singlet oxygen can,
indeed, be produced and optically detected upon two-photon
excitation of either a substituted difuranonaphthalene or a
substituted distyryl benzene in a focused laser beam. Properties
of these molecules pertinent to the photosensitized production
of singlet oxygen have also been examined.
In certain molecules, the production of an excited-state singlet
can also occur upon the simultaneous absorption of two lower-
energy photons.2,3 Of particular interest is the case where the
transition proceeds via a virtual state, following selection rules
that differ from those for a one-photon transition.2 A range of
phenomena derived from such nonlinear two-photon transitions
can be studied using pulsed lasers that deliver high photon
intensities.3 Of special interest is the opportunity to selectively
excite a two-photon transition at the point of a focused laser
beam where a local domain of sufficiently high fluence can be
obtained. This aspect of pumping a two-photon transition can
be used to impart spatial resolution to an experiment and thus
is pertinent, for example, in the construction of emission
microscopes.4,5 Moreover, irradiation at a wavelength that does
not coincide with the dominant one-photon transition facilitates
depth penetration in samples that might otherwise be opaque.
* To whom correspondence should be addressed.
† Aarhus University.
‡ Risø National Laboratory.
(4) Williams, R. M.; Piston, D. W.; Webb, W. W. FASEB J. 1994, 8,
804-813.
(1) Singlet Oxygen; Frimer, A. A., Ed.; CRC Press: Boca Raton, FL,
1985; Vols. I-IV.
(2) McClain, W. M. Acc. Chem. Res. 1974, 7, 129-135.
(3) Kershaw, S. In Characterization Techniques and Tabulations for
Organic Nonlinear Optical Materials; Kuzyk, M. G., Dirk, C. W., Eds.;
Marcel Dekker, Inc.: New York, 1998; pp 515-654.
(5) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10763-10768.
(6) Fisher, W. G.; Partridge, W. P.; Dees, C.; Wachter, E. A. Photochem.
Photobiol. 1997, 66, 141-155.
(7) Fisher, W. G.; Lytle, F. E. Anal. Chem. 1993, 65, 631-635.
10.1021/ja003468a CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/19/2001

1216 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Frederiksen et al.
Chart 1
Figure 1. Schematic diagram of the optical layout used for two-photon
irradiation.
Experimental Section
Apparatus and Techniques. Although most of the work was done
using a nanosecond laser system, a few experiments were performed
with a femtosecond system.
band Xe lamp output. Spectral discrimination of the absorption signals
was obtained with a Chromex model 250is spectrograph. Signals were
monitored with either a gated intensifier/CCD camera (Princeton
Instruments) or a photomultiplier tube (Hamamatsu R928).
The femtosecond experiments used a regenerative amplified Ti:
sapphire system (Clark MXR CPA 1000) operated at a repetition rate
of 1 kHz. Samples were irradiated at 802 nm with 90 fs pulses (fwhm)
at energies over the range ∼1-50 µJ/pulse. The laser output (∼2 mm
diameter beam) was focused into the center of a 4 cm path length sample
cell using a 20 cm focal length lens. Thus, upon entering and exiting
the cell, the laser beam had a diameter of ∼200 µm.
In the two-photon experiments using the difuranonaphthalenes Ia-
e, most of the data were recorded using sensitizer concentrations of 2
× 10-3 M in air-saturated toluene. For the experiments in which data
from Id were compared to data from compound II, a concentration of
3 × 10-4 M was used. All of the data on compound II were recorded
using a concentration of 3 × 10-4 M. In the one-photon experiments,
the sensitizer concentration was adjusted to yield a ground-state
absorbance of ∼0.5 at the irradiating wavelength. For all sensitizers,
the clear onset of one-photon ground-state absorption (i.e., A > ∼0.02)
occurred at wavelengths shorter than ∼520 nm. Most importantly, for
the samples used in the two-photon experiments, measured absorbances
were <0.002 in the range ∼550-800 nm (note that some light scattering
also contributes to the absorbances measured). Absorption spectra were
recorded with either a Cary model 17 spectrophotometer or a Hewlett-
Packard model 8453 diode array spectrometer.
Sensitizer Preparation. The difuranonaphthlenes Ia-e and the
distyrylbenzene II used as singlet oxygen sensitizers are shown in Chart
1. The syntheses of these compounds are outlined in Schemes 1 and 2.
Briefly, K2CO3-promoted substitution of either 1-phenyl-2-bromode-
canone or 1-(4-bromophenyl)-2-bromodecanone with 1,5-dihydroxy-
naphthalene produced the intermediary 1,5-bis(1-arylnonan-1-on-2-
yloxy)naphthalenes. Treatment of the latter with methanesulfonic acid
in methylene chloride yielded the 3,8-diaryl-2,7-dioctyldifurano[2,3-
a:2′,3′-f]naphthalenes Ia and Ic, respectively. Compound Ic was treated
with copper(I) cyanide under Ullman-type conditions to give the dinitrile
Ib. Alternatively, bromine-to-lithium exchange followed by reaction
with dimethylformamide produced the dialdehyde Id. Compound Ie
was prepared from Ia by benzoylation in a Friedel-Crafts reaction.
The meta substitution observed appears to result from prior complex-
ation of the Lewis acid, AlCl3, to the oxygen heterocycle. Details of
these respective syntheses are provided elsewhere.8
2,5-Bis(4-diphenylaminostyryl)-1,4-dibromobenzene (II) was syn-
thesized via a Horner-Wadsworth-Emmons reaction between [2,5-
dibromo-4-(diethoxyphosphorylmethyl)benzyl]phosphonic acid diethyl
ester and 4-(diphenylamino)benzaldehyde.9 The phosphonate ester was
prepared from 2,5-dibromo-1,4-xylene,9 and the 4-(diphenylamino)-
benzaldehyde was prepared via formylation of triphenylamine.10 In the
coupling reaction to make II, 251 mg (0.47 mmol) of the phosphonate
ester and 275 mg (1.0 mmol) of the carboxy triphenylamine were
dissolved in 10 mL of DMF under an atmosphere of argon. Potassium
tert-butylate (200 mg, 1.78 mmol) was added, and the color of the
mixture immediately changed from light yellow to dark red, and the
orange-yellow product began to precipitate. The reaction mixture was
stirred at ambient temperature for 1 h and then diluted with water, and
The nanosecond experiments were performed using a Nd:YAG-
pumped optical parametric oscillator (OPO) as the irradiation source
(Quanta-Ray GCR 230 and MOPO 710, pulse fwhm ∼5 ns, 10 Hz
repetition rate). In some of the studies, specific irradiation wavelengths
were also obtained by (1) stimulated Raman scattering of the Nd:YAG
output in a high-pressure cell of H2 and (2) frequency doubling of the
OPO output. For the one-photon experiments, the sample was irradiated
with an unfocused beam ∼3 mm in diameter using discrete laser
wavelengths less than ∼400 nm. A 1 cm path length cuvette was used
for these studies. For the two-photon experiments, discrete laser
wavelengths in the range ∼590-880 nm were used, and the laser output
was focused in the sample using a 35 mm focal length lens. Under the
latter conditions, which are far from optimal,4 a beam width <100 µm
is obtained at the focus. A 4 cm path length cuvette was used for the
two-photon studies, and the laser beam focus was centered in this
cuvette. The volume of sample used was typically 3 mL. A diagram of
the optical layout used for the two-photon experiment is shown in Figure
1. In this case, two optical paths were created using a prism beam-
splitter. The polarization of the beam in one path was then chosen to
be either parallel or perpendicular to that in the other beam path using
a zero-order λ/2 phase retardation plate (Alphalas). The separate beams
were then focused into the sample using the same 35 mm focal length
lens. We established that, in this configuration, the respective beams
properly intersected at the same point in the sample by comparing the
intensity of the singlet oxygen phosphorescence signal thus obtained
to that observed from a single beam configuration. Specifically, in the
two-beam configuration with identical polarization, the signal intensity
observed was identical to that observed upon focusing a single beam
of comparable energy into the sample.
For the two-photon experiments, the laser intensity, IL, was varied
over the range ∼1-30 mJ pulse-1 cm-2 using neutral density filters
placed in the unfocused beam (∼3 mm diameter). For the one-photon
experiments, IL was varied by changing the output energy of the flash
lamps used to pump the Nd:YAG laser. Laser energies were quantified
using volume calorimeters (Scientech).
Singlet oxygen phosphorescence was monitored with a North Coast
model EO-817P Ge detector operated at 77 K. The 1270 nm singlet
oxygen emission was detected through a silicon window (CVI optics)
and an interference filter (Barr Associates, fwhm ) 50 nm). Signals
were recorded and averaged using a Tektronix model TDS754A digital
oscilloscope. Time-resolved absorption measurements were recorded
using a 150 W steady-state Xe lamp as the probe source. A chopper
(New Focus model 3501) synchronized to the timing of the pulsed
excitation laser limited the exposure time of the sample to the broad
(8) Jørgensen, M.; Krebs, F. C.; Bechgaard, K. J. Org. Chem. 2000, 65,
8783-8785.
(9) Blum, J.; Zimmerman, M. Tetrahedron 1972, 28, 275-280.
(10) Baker, T. N.; Doherty, W. P.; Kelley, W. S.; Newmeyer, W.; Rogers,
J. E.; Spalding, R. E.; Walter, R. I. J. Org. Chem. 1965, 30, 3714-3718.

Two-Photon Photosensitized Production of Singlet Oxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1217
Scheme 1
Scheme 2
Table 1. Singlet Oxygen Yields and Two-Photon Cross Sections
for Compounds Ia-d and IIa
the product was filtered. The solid material was recrystallized from
benzene to give 240 mg (66% yield) of an orange microcrystalline
powder which decomposed at 275 °C. H NMR (CDCl3, 250.1 MHz)
δ: 7.05-7.2 (m, 16H), 7.24-7.38 (m, 12H), 7.45 (d, 4H, J ) 9 Hz),
7.87 (s, 2H) ppm. 13C NMR (CDCl3, 62.9 MHz) δ: 123.3, 123.5, 123.8,
124.3, 125.2, 128.3, 129.8, 130.4, 130.9, 131.9, 137.7, 147.8, 148.5
ppm.
1
two-photon absorption
one-photon singlet
oxygen quantum yield
cross section at 618 nmb
sensitizer
(×1050 cm4 s photon-1
)
Ia
Ib
Ic
Id
II
0.36 ( 0.04
0.36 ( 0.04
0.42 ( 0.05
0.49 ( 0.06
0.46 ( 0.05
8 ( 2
139 ( 35
7 ( 2
205 ( 50
Results and Discussion
Using guidelines that are available for maximizing two-photon
absorption cross sections,2,11 we set out to prepare efficient two-
photon singlet oxygen sensitizers. The molecules examined in
this regard, the difuranonaphthalenes Ia-e, are shown in Chart
1. The pendant n-octyl moieties were included simply to increase
the solubility of these compounds in toluene, the solvent in
which our work was done. Albota et al.11 have recently shown
that substituted distyryl benzenes can have appreciable two-
photon absorption cross sections. Although we have determined
that singlet oxygen yields from this general class of molecules
can be low upon one-photon irradiation,12 Albota et al.
speculated that one distyryl benzene in particular, the bis-
a Data recorded in toluene. b Obtained by comparing two-photon
singlet oxygen phosphorescence intensities, normalized by the one-
photon singlet oxygen yield, to the two-photon singlet oxygen
phosphorescence intensity from compound II, again normalized by the
one-photon singlet oxygen yield (see text). The errors shown were
obtained by combining our experimental error (∼(10%) with the error
reported for our reference standard ((15%). Data from Ia-d were
recorded at 17 mJ pulse-1 cm-2, whereas data from II were recorded
at 13 mJ pulse-1 cm-2
.
diphenylamino compound II (Chart 1), might, in fact, be a good
two-photon singlet oxygen sensitizer. We thus also examined
the behavior of II as a singlet oxygen sensitizer.
One-Photon Results. We ascertained that Ia-d and II
generate singlet oxygen in appreciable yield upon one-photon
excitation (Table 1). In these experiments, the singlet oxygen
yields were quantified by comparing the intensity of the 1270
nm singlet oxygen phosphorescence signal obtained upon
irradiation of Ia-d and II to the singlet oxygen signal observed
(11) Albota, M.; Beljonne, D.; Bre´das, J.-L.; Ehrlich, J. E.; Fu, J.-Y.;
Heikal, A. A.; Hess, S. E.; Kogej, T.; Levin, M. D.; Marder, S. R.; McCord-
Maughon, D.; Perry, J. W.; Rockel, H.; Rumi, M.; Subramaniam, G.; Webb,
W. W.; Wu, X.-L.; Xu, C. Science 1998, 281, 1653-1656.
(12) Dam, N.; Scurlock, R. D.; Wang, B.; Ma, L.; Sundahl, M.; Ogilby,
P. R. Chem. Mater. 1999, 11, 1302-1305.

1218 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Frederiksen et al.
ically, upon irradiation of compound II, singlet oxygen lifetimes
in the range ∼27-29 µs were recorded. Upon irradiation of Ib
and Id, slightly shorter lifetimes were obtained (∼18-20 µs).
The latter data reflect quenching of singlet oxygen by the
sensitizer, an observation confirmed in independent one-
photon experiments. The singlet oxygen lifetimes observed were
independent of IL over the range 1-30 mJ pulse-1 cm-2. Upon
prolonged irradiation (i.e., ∼2000 laser pulses) of Ia-e at 10
mJ pulse-1 cm-2 in an air-saturated solution with the laser
focused into a sample volume of 3 mL, the ground-state
absorption spectra showed changes that were consistent with
the degradation of ∼10% of the total amount of sensitizer
present in the cuvette.
Figure 2. Plot of the singlet oxygen phosphorescence intensity, I∆,
against the intensity of the irradiation laser, IL. Data were recorded
upon irradiation of Ib in toluene at 355 nm (one-photon pumping
scheme). The dashed line results from a linear fit to the first seven
data points.
A number of control experiments were also performed.
Specifically, singlet oxygen signals were not observed from
sensitizer-free toluene upon irradiation over this same wave-
length range of ∼590-880 nm with a focused laser. We also
irradiated two “standard” molecules well known to be efficient
one-photon singlet oxygen sensitizers, acridine and pyrene, using
a focused laser beam at wavelengths approximately twice those
of the corresponding one-photon transitions. Singlet oxygen
signals were not observed upon irradiation of pyrene over the
range ∼600-670 nm and of acridine at 683 nm.
upon irradiation of acridine. Experiments were performed in
air-saturated toluene using a reference singlet oxygen quantum
yield of 0.83 ( 0.06 for acridine.13 Samples were irradiated at
355 nm with energies <5 mJ pulse-1 cm-2, and the data were
normalized for the number of photons absorbed by the respective
sensitizers.
For two-photon absorption, I∆ should scale according to the
square of IL. Using compound Ib, the data, indeed, meet this
expectation at low IL (Figure 3). However, as with the one-
photon experiments, the I∆ data recorded at higher laser
intensities do not increase as much as expected on the basis of
this quadratic dependence (Figure 3). On the basis of our one-
photon control experiments (vide supra), we infer that this
curvature likewise reflects a decrease in the yield of the
sensitizer triplet state, T1, with an increase in IL. Specifically,
after two-photon excitation, secondary absorption by a sensitizer
excited state within the irradiating pulse is presumed to initiate
a process that results in a smaller yield of T1. This secondary
absorption could either be a one- or two-photon transition.
Again, because T1 is the immediate precursor to singlet oxygen,
any event that results in a decrease in the T1 yield will be
reflected in the yield of singlet oxygen. In this regard, it is
pertinent to note that, in two-photon fluorescence studies,
significant deviations from a quadratic dependence on IL are
frequently observed.16 The latter data have been attributed to a
variety of factors, including secondary absorption by an excited
state.16 For our present study, and arguably most importantly,
the plot of I∆ against IL shown in Figure 3 indicates that the
singlet oxygen data recorded upon ∼590-680 nm irradiation
with a focused laser are certainly not consistent with a one-
photon pumping scheme.
Upon sensitizer excitation in a one-photon pumping scheme,
the intensity of the singlet oxygen signal, I∆, should increase
linearly with an increase in the intensity of the pump laser, IL.
Using compound Ib, we ascertained that this was, indeed, the
case at low laser intensities (<7 mJ pulse-1 cm-2 at 355 nm).
At higher laser intensities, however, I∆ did not increase as much
as expected on the basis of this linear correlation, and plots of
I∆ against IL showed curvature concave to the IL axis (Figure
2). Similar observations have been made for the production of
singlet oxygen in other systems.14 These latter data have been
attributed to secondary absorption of light by a sensitizer excited
state within the irradiating pulse at high IL that, in turn, decreases
the relative yield of the sensitizer triplet state.14 Independent
transient absorption experiments of our sensitizers support this
interpretation. Specifically, upon irradiation of compound Ib
at 355 nm, we recorded a transient absorption band with a
maximum at ∼620 nm that we assign to the Ib triplet state on
the basis of an oxygen quenching experiment (quenching rate
constant ∼3 × 109 s-1 M-1). In plots of this transient absorbance
against IL, the data likewise showed curvature concave to the
IL axis. Moreover, this curvature was identical to that seen in
the plots of I∆ against IL, which is to be expected if the sensitizer
triplet state is a precursor to singlet oxygen.
Nanosecond Two-Photon Results. Upon irradiation of Ia-e
at discrete wavelengths over the range ∼590-680 nm and upon
irradiation of II at discrete wavelengths over the range ∼780-
880 nm, we were able to detect the characteristic time-resolved,
1270 nm singlet oxygen phosphorescence from the laser beam
focal point. For compounds Id and II, singlet oxygen signals
could be detected with laser intensities, IL, as low as ∼1 mJ
pulse-1 cm-2. The signal decay was always single exponential,
and the singlet oxygen lifetimes obtained were consistent with
those expected for experiments performed in toluene.15 Specif-
Action spectra were recorded for Ib and Id by plotting I∆,
normalized with respect to irradiation intensity, against the
irradiation wavelength over the range 590-670 nm (Figures 4
and 5). An analogous action spectrum was recorded for II over
the range ∼780-880 nm (Figure 6). For Ib and II, the data
show a distinct absorption band at approximately twice the
wavelength of the one-photon absorption bands. For Id,
however, the action spectrum does not reveal such a distinct
band over the wavelength range examined. For many molecules,
two-photon fluorescence excitation spectra can be recorded and,
in turn, can similarly be compared to the one-photon absorption
(13) Wilkinson, F.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref.
Data 1993, 22, 113-262.
(14) Gorman, A. A.; Hamblett, I.; Lambert, C.; Prescott, A. L.; Rodgers,
M. A. J.; Spence, H. M. J. Am. Chem. Soc. 1987, 109, 3091-3097.
(15) Wilkinson, F.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref.
Data 1995, 24, 663-1021.
(16) Xu, C.; Webb, W. W. In Topics in Fluorescence Spectroscopy;
Lakowicz, J., Ed.; Plenum Press: New York, 1997; Vol. 5, Nonlinear and
Two-Photon-Induced Fluorescence, pp 471-540.

Two-Photon Photosensitized Production of Singlet Oxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1219
Figure 5. Plot of the normalized singlet oxygen phosphorescence
intensity, I∆, against the irradiation wavelength for Id (top and right
axes). Superimposed on this plot is the one-photon absorption spectrum
of Id (bottom and left axes).
Figure 3. (a) Plot of the singlet oxygen phosphorescence intensity,
I∆, against the intensity of the irradiation laser, IL. Data were recorded
upon irradiation of Ib in toluene with a focused laser at 618 nm.
Functions that illustrate the ideal one-photon (i.e., linear) as well as
two-photon (i.e., quadratic) dependence of I∆ against IL are also shown.
On the basis of material in Figure 2, plots of I∆ against IL for a one-
photon pumping scheme are expected to be linear only at low values
of IL. Thus, to approximate the behavior expected for a one-photon
process, a linear fit was applied to the first three data points only (dashed
line). The solid line is a quadratic fit to the first six data points. (b)
Logarithmic plot of the data shown in (a). The solid line is a linear fit
to the first five data points (the 0,0 point in (a) must be excluded from
the logarithmic plot) and has a slope of 1.9 ( 0.2. A slope of 2.0 is
expected for a two-photon process.
Figure 6. Plot of the normalized singlet oxygen phosphorescence
intensity, I∆, against the irradiation wavelength for II (top and right
axes). Superimposed on this plot is the one-photon absorption spectrum
of II (bottom and left axes).
profiles likely reflect structure-dependent changes in the number
of final states that can be accessed via a virtual state.
Although the preceding data are consistent with the produc-
tion of singlet oxygen via a two-photon S0 f Sn transition in
the sensitizers Ia-e and II, it is important, for completeness,
to consider other possible mechanisms of singlet oxygen
production. For example, upon ∼590-880 nm irradiation of
our samples, singlet oxygen might be produced (a) from a
charge-transfer (CT) state formed between oxygen and the
abundant solvent molecules,20 (b) upon an oxygen-enhanced S0
f Tn transition in the sensitizer,21 and/or (c) upon excitation
of oxygen itself.22 These alternative mechanisms can be
discounted, however, on the basis of the following points. First,
the induced S0 f Tn transition is normally observed at oxygen
pressures significantly greater than 1 atm, not in air-saturated
samples at 1 atm.21 Second, if singlet oxygen were produced
from an oxygen-toluene CT state or from a direct transition in
oxygen, signal would have been observed in all instances,
including from the sensitizer-free solvent. We thus conclude
that our data are the result of a two-photon S0 f Sn pumping
scheme in Ia-e and II.
Figure 4. Plot of the normalized singlet oxygen phosphorescence
intensity, I∆, against the irradiation wavelength for Ib (top and right
axes). Superimposed on this plot is the one-photon absorption spectrum
of Ib (bottom and left axes).
spectra. From such studies, it is clear that, in some cases, the
spectral profile of one-photon absorption can differ significantly
from that of two-photon absorption at twice the wavelength.5,16
Such data are consistent with those of Id shown in Figure 5.
Nevertheless, there is also ample evidence to indicate that, as
with our data from Ib and II, the one-photon absorption profile
can resemble the two-photon profile at twice the wavelength
(e.g., fluorescence excitation data recorded for â-naphthol,
benzene, toluene, and fluorescein, among other molecules).5,7,16-19
Among other things, these differences in two-photon absorption
(17) Chen, C. H.; McCann, M. P. J. Chem. Phys. 1988, 88, 4671-4677.
(18) Birge, R. R. Acc. Chem. Res. 1986, 19, 138-146.
(19) Birge, R. R. In UltrasensitiVe Laser Spectroscopy; Kliger, D. S.,
Ed.; Academic Press, Inc.: New York, 1983; pp 109-174.
(20) Scurlock, R. D.; Ogilby, P. R. J. Phys. Chem. 1989, 93, 5493-
5500.
(21) Evans, D. F. J. Chem. Soc. 1957, 1351-1357.
(22) Evans, D. F. Chem. Commun. 1969, 367-368.

1220 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Frederiksen et al.
The singlet oxygen signals recorded upon two-photon ir-
radiation of Ia-e and II were significantly smaller than signals
typically recorded in a one-photon pumping scheme. For
example, upon one-photon excitation of Ib with 355 nm
irradiation at <1 mJ pulse-1 cm-2, singlet oxygen signal-to-
noise ratios of ∼15 can routinely be recorded in our system
using a single laser pulse. However, to record a signal-to-noise
ratio of ∼15 in the two-photon experiment where IL ) 30 mJ
pulse-1 cm-2 typically required the summation of data from
∼1000 laser pulses. Moreover, in the two-photon experiments,
it was necessary to discriminate between the singlet oxygen
signal and a signal that originates as a consequence of the
interaction between the solvent and the radiation field in the
focused laser (e.g., Raman scattering and supercontinuum
emission23). In some cases, the latter signal can be sufficiently
large as to saturate our detector. If the detector does not saturate,
however, the decay of the undesired signal is limited by the
response time of our system (∼400 ns) and can be distinguished
from the more slowly decaying singlet oxygen signal. The
magnitude of this background signal at 1270 nm depends both
on the solvent and on the irradiation wavelength. For example,
this signal generally does not cause problems when focusing
∼600-800 nm light into D2O and toluene. On the other hand,
in CCl4 and CS2, the undesired signal can be appreciable.
Nanosecond Two-Photon Absorption Cross Sections.
Several experiments were done to assess the absolute as well
as relative two-photon absorption cross sections in Ia-d. To
describe this work, recall that both one- and two-photon
pumping schemes will ultimately result in the formation of the
lowest excited singlet state of the sensitizer, S1. Under conditions
that preclude or minimize secondary absorption of light by the
sensitizer within the irradiating pulse (i.e., low IL, vide supra),
the probability with which subsequent events occur will be
independent of the mechanism by which S1 was formed.
Specifically, the efficiency of S1 f T1 intersystem crossing and
the efficiency with which ground-state oxygen quenches the
sensitizer excited states to yield singlet oxygen will be identical
in both the one- and two-photon pumping schemes. Thus, by
normalizing the relative singlet oxygen intensities, I∆, obtained
from the two-photon experiments by the corresponding one-
photon singlet oxygen yield (Table 1), one should obtain a
parameter proportional to the two-photon absorption cross
section.
Ib and Id). Thus, two-photon singlet oxygen signals recorded
upon 800 nm irradiation of II must be compared to two-photon
singlet oxygen signals recorded upon irradiation of Ia-d at a
different wavelength and the data corrected for the respective
photon energies. For solutions of Id and II, both at 3 × 10-4
M, we find that Id has a two-photon absorption cross section
at 618 nm that is ∼2.2 times smaller than that of II at 800 nm.
Relative 618 nm two-photon absorption cross sections in Ia-
d, independently obtained, were then scaled accordingly (Table
1). Note that the smallest cross sections thus obtained, (7-8)
× 10-50 cm4 s photon-1 for Ia and Ic, are larger than the lower
limit estimated for the cross sections using acridine (vide supra).
The data in Table 1 indicate that the two-photon absorption
cross sections for Ia-d are reasonably large and depend
significantly on the nature of the substituent attached to the
chromophore.
It is well established that two-photon absorption probabilities
can depend on the relative polarization of the two photons that
are simultaneously absorbed by the molecule.2,19 Such a
polarization dependence, however, is not a requirement of a
two-photon transition. In our experiment, we are able to control
the relative polarization of the pumping photons such that their
polarization axes are either parallel or perpendicular to each
other (Figure 1). For all compounds examined, however, the
intensities of the singlet oxygen signals observed upon irradia-
tion were independent of the relative polarization of the light
in the two irradiating optical paths.
Femtosecond Two-Photon Results. All of the experiments
discussed thus far have been performed using a nanosecond laser
as the irradiation source. It is well documented, however, that
for two-photon experiments, there are many advantages to the
use of a femtosecond laser as the irradiation source.6 The
principal attribute of a much shorter irradiation pulse is the
ability to deliver a comparatively high peak power and fluence
using a comparatively small incident energy. Thus, two-photon
transitions can be more readily pumped with significantly less
photoinduced damage incurred by the system. Moreover, the
repetition rate of a femtosecond system can be much greater
than that of a typical nanosecond system, thus facilitating the
process of signal averaging and the detection of weak emission
signals. We thus felt it was important, in the least, to establish
that singlet oxygen could likewise be detected in a two-photon
photosensitized process using a femtosecond laser as the
irradiation source.
Indeed, upon irradiation of compound II with a femtosecond
laser at 802 nm in toluene (∼1-50 µJ/pulse), we were readily
able to observe singlet oxygen phosphorescence in a time-
resolved experiment. As in the nanosecond experiments, it was
necessary to discriminate between the singlet oxygen signal and
a background signal arising from the laser-solvent interaction.
Nevertheless, this was easily done, and appreciable singlet
oxygen signal-to-noise levels (>20) were rapidly obtained (<5
s) at a sampling frequency of 100 Hz. These data are consistent
with the previously mentioned advantages of using a femto-
second laser to pump a two-photon transition.
First, we note the absence of singlet oxygen signals upon
∼670-690 nm irradiation of pyrene and acridine, for which
two-photon cross sections of 0.22 × 10-50 and 2.0 × 10-50
cm4 s photon-1, respectively, have been measured at 694 nm.3
These values, which are small in comparison to values for
molecules considered to be efficient two-photon absorbers,11
allow us to estimate a lower limit for the two-photon cross
sections in Ia-d. However, a better assessment of the two-
photon absorption cross sections in Ia-d can be made by using
compound II as a reference standard, for which a value of
(450 ( 70) × 10-50 cm4 s photon-1 at 800 nm has been
reported.11
As shown in Figure 6, we find that compound II has a two-
photon absorption maximum at ∼800 nm. Unfortunately,
compound II does not have an appreciable two-photon cross
section at ∼618 nm, for example, where Ib shows a nice
maximum (Figure 4). Moreover, compounds Ib and Id do not
have an appreciable two-photon absorption at 800 nm (i.e.,
singlet oxygen was not observed upon 800 nm irradiation of
Conclusions
We have demonstrated that time-resolved singlet oxygen
phosphorescence can be detected from the small spatial domain
of a focused laser beam after the two-photon excitation of a
photosensitizer. In this work, a series of reasonably efficient
two-photon singlet oxygen sensitizers were prepared. The singlet
oxygen signals observed reflect the unique features of this
nonlinear optical experiment: (1) depth penetration in the
(23) The Supercontinuum Laser Source; Alfano, R. R., Ed.; Springer-
Verlag: New York, 1989.

Two-Photon Photosensitized Production of Singlet Oxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1221
sample afforded by irradiation in a spectral region void of the
more dominant one-photon linear transitions and (2) spatial
resolution afforded by irradiation with a focused laser beam.
Thus, our results are significant with respect to two-photon
pumping schemes in photodynamic therapy, a procedure where
singlet oxygen can be a key reactive intermediate.24 Our results
are also pertinent with respect to the construction of a two-
photon emission microscope that can be used to create singlet
oxygen images of heterogeneous materials.
Acknowledgment. This work was supported by grants from
the Danish Natural Science Research Council and from the
Council’s initiative on Materials Research. The authors thank
Søren Keiding and Jan Thøgersen for the use of, and assistance
with, the femtosecond laser system.
(24) Fuchs, J.; Thiele, J. Free Rad. Biol. Med. 1998, 24, 835-847.
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