September 1, 2004 / Vol. 29, No. 17 / OPTICS LETTERS
Enhanced two-photon fluorescence excitation by resonant
grating waveguide structures
ICFO—Insitut de Ciències Fotòniques and Universitat Politècnica de Catalunya, 08034 Barcelona, Spain
T. Katchalski, E. Teitelbaum, and A. A. Friesem
Department of Physics of Complex Systems, Weizmann Institute of Science, 76100 Rehovot, Israel
Laser Laboratorium Göttingen e.V., Hans-Adolf-Krebs-Weg-1, 37077 Göttingen, Germany
Received January 28, 2004
Enhanced two-photon fluorescence (TPF) spectroscopy with novel high-finesse resonant polymeric grating
waveguide structures (GWSs) is presented.Under resonant conditions the field enhancement at the surface
of a GWS can be exploited for TPF spectroscopy without the need for highly focused laser excitation light.
We compare the TPF obtained by placing a drop of tetramethylrhodamine (TMR) on top of a GWS with that
obtained with TMR on top of a glass substrate. Our procedure and results indicate that the detection of TPF
can be improved by a factor of 10 with resonant GWSs.
© 2004 Optical Society of America
Two-photon excitation performed with near-infrared
radiation has a number of advantages compared with
(1) low scattering because of the large energy gap
between excitation and emission radiation, (2) re-
duction of static photobleaching of the dyes that are
used because there is a quadratic dependence of the
absorption on intensity,1,2(3) greater tolerance of cells
and tissue to near-infrared radiation, and (4) less
Conventional two-photon excitation, however, re-
quires highly intense, focused laser light, so the
photodamage threshold is lowered.3
required high instantaneous photon flux densities
??1031photons?cm2s?2and yet avoid tight focusing,
we resort to low-loss high-finesse polymeric resonant
grating waveguide structures (GWSs).4
configuration of such a GWS consists of a substrate, a
waveguide layer, and a grating layer on top.
the GWS is illuminated with an ultrashort light
pulse, most of the light is directly transmitted through
the structure and some is diffracted by the grating,
trapped in the waveguide layer, and subsequently par-
tially rediffracted outward.
and angular orientation of the incident beam, the GWS
resonates, where the rediffracted beam destructively
interferes with the transmitted beam and most of the
incident light is reflected.
occurs in the spectrum of the illuminating pulse.5
For our purposes the most important feature of a
GWS is the large optical field enhancement that can
be achieved at the grating’s surface, despite the low
coupling efficiency.Thus the GWS can be exploited
for two-photon fluorescence (TPF) excitation of a layer
that is deposited on top of the grating layer.
have a number of additional attractive features for TPF
applications. In particular, they are compact and ro-
To achieve the
At a specific wavelength
Specifically, a sharp dip
bust to simplify experimental setups, and they are rela-
tively easy to fabricate so they can readily be incor-
porated intowidespread biological
In recent years there have been some attempts to
enhance TPF by use of resonant phenomena such as
surface plasmon resonance on smooth silver films6and
on silver nanoparticle fractal clusters.7
though in both cases the performance is excellent, the
technical requirements and costs are high, and, for
clusters, special dyes must be synthesized.
To evaluate the efficacy of our GWS we performed
several experiments.First we determined the reso-
nant behavior of the GWS with and without a drop
of tetramethylrhodamine (TMR) on top.
GWS with the TMR drop, we compared the TPF inten-
sity emitted on and off resonance; the TPF intensity
was measured as a function of wavelength at a fixed
polarization and as a function of polarization at a fixed
wavelength.Finally, we compared the TPF intensity
when a GWS was used with that obtained when a 90±
quartz prism in total internal reflection or glass sub-
strate or a waveguide layer was used.
Several GWSs of 1-cm diameter were fabricated.
We used optical quality glass substrates onto which
the waveguide and the photoresist layers were spin
coated.The waveguide consisted of a polyimide layer
of approximately 430-nm thickness (refractive index,
n ? 1.7).The grating was holographically recorded
into a Shipley S1805 photoresist layer.
grating period was ?523 nm and the grating thickness
was 450 nm. In principle, a decrease in the grating
thickness narrows the resonance bandwidth, whereas
a change in the grating period shifts the resonance
wavelength.The grating structure was optimized to
minimize surface roughness, with different exposures,
development procedures, and heat treatments.
Then, for the
0146-9592/04/171989-03$15.00/0 © 2004 Optical Society of America
OPTICS LETTERS / Vol. 29, No. 17 / September 1, 2004
waveguide layer also served as a stopping layer for the
wet-etching development of the grating layer and thus
assisted in achieving a high uniformity.
the nonlinearity of the etching process the resultant
grating had a square profile with an approximately
40% duty cycle.4
Afterward a drop of 15-mM TMR
solution in milli-Q water (pH 7.5) was deposited on
top of the GWS. After evaporation of the solvent,
the TMR molecules remained immobile on the surface
of the GWS.The presence of this TMR layer should
lead, according to rigorous coupled-wave analysis, to a
shift of approximately 10 nm in the resonance wave-
length as a result of the change of index of refraction.
The experimental arrangement for evaluating TPF
together with a GWS configuration is shown schemati-
cally in Fig. 1.In our experiments the excitation
light was derived from a mode-locked Ti:sapphire laser
(Coherent) operated at a frequency of 76 MHz.
pulse width was 150 fs and the spectral bandwidth
was ?8 nm. The wavelength of the excitation light
could be tuned from 690 to 980 nm but was generally
centered at the resonant wavelength of the GWS.
GWS was mounted upon a positioning stage (Physik
Instrumente), which allowed for transverse transla-
tion and rotation to ensure the normal incidence for
which the GWS was designed.
the angular alignment tolerances of the incident beam
The incident laser beam of 21-kW
peak power was slightly focused to a 500-mm beam
waist. TPF occurs at the location of the excitation
beam. The TPF was collected by a spherical lens
(f ? 50 mm, N.A. ? 0.8), focused onto the entrance
slit of a spectrometer (Jobin Yvon), and detected by
a backthinned CCD linear array (Hamamatsu). The
excitation light of the Ti:sapphire laser was blocked
by a near-infrared filter (BG39, Schott) placed in front
of the entrance slit of the spectrometer.
Some representative experimental results are shown
in Figs. 2 and 3. Figure 2 shows the transmitted
spectral response of the bare (without TMR) GWS, the
GWS with TMR, and the incident pulse (reference).
The GWS was illuminated at normal incidence with
transverse electric (TE) polarization, in which the
electric field of the incident light is parallel to the
grating grooves.As is evident from these experimen-
tal results, the transmitted intensities do not decrease
to zero, indicating that our GWS has some scatter loss.
Also, the resonance peak of the GWS with TMR was
shifted by 9 nm from that of the bare GWS.
transverse magnetic (TM) polarization illumination
the resonance peak was shifted by 10 nm to a shorter
wavelength. As is evident, the transmission’s spec-
tral profile has a dip at the resonant wavelength of
lres? 844 nm with a FWHM of 2 nm for TE polariza-
tion excitation. For the TM polarization at the same
wavelength, i.e., out of resonance, the spectrum of the
transmitted light corresponded to the full spectrum of
the incident pulse.
Figure 3 shows TPF as a function of wavelength
for several excitation wavelengths obtained with a
GWS onto which TMR has been deposited.
measurements were made at each wavelength.
ensure that the fluorescence is indeed due to the GWS
At normal incidence,
enhancement we first varied the incident laser wave-
length from 830 to 856 nm at fixed polarization and
then varied the polarization at fixed wavelengths.
is evident from Fig. 3, there is very low background
TPF away from the resonance.
the TPF intensity increases strongly, reaching its
maximum at 844 nm, indicating strong field enhance-
ment.The peak intensity is approximately ten times
ration with a TMR drop.
(a) Experimental arrangement, (b) GWS configu-
ND, neutral-density filter; L1,
response of a polymeric GWS for TE-polarization excita-
tion: triangles, without TMR; squares, with TMR; solid
curve, reference pulse.For a bare GWS the transmission
intensity drops by 90%.
Experimental measurements ofthe spectral
at resonance (TE mode); open squares, the excitation wave-
length out of resonance (TM mode); triangles, maximum
two-photon absorption (TE mode); circles, lower limit of the
FWHM (TE mode). Average intensity, 127 W?cm2.
TPF signal with GWS for several excitation wave-
Filled squares denote the excitation wavelength
September 1, 2004 / Vol. 29, No. 17 / OPTICS LETTERS Download full-text
TPF signal for l ? 844 nm.TIRF, total internal
higher than the background.
two-photon absorption of TMR, lmax ? 849 nm, the
fluorescence signal was reduced by a factor of 2.
For wavelengths at the FWHM of the resonance
bandwidth, the measured fluorescence decreased by
approximately 20%. These results indicate that the
maximum TPF intensity is observed only under reso-
nant conditions.We also compared the experimental
TPF results with the GWS at resonance to those with
a prism onto which TMR was deposited.
are presented in Fig. 4.As is evident, the results
with the GWS are better by at least a factor of 3.
Finally, we performed measurements of TPF after
the GWS was replaced with a substrate and a wave-
guide layer. The measured TPF values were compa-
rable to the low-background values obtained away from
resonance condition. This result indicates that the
TPF enhancement with the GWS is an order of mag-
nitude higher. No TPF was detected in the spectral
region from 350 to 500 nm or in regions of the GWS
Close to the maximum
without an immobilized TMR.
clude other nonlinear effects such as surface second-
To conclude, our procedure and results indicate that
the detection of TPF can indeed be more sensitive with
a resonant GWS.We expect that the overall detection
sensitivities will increase even further as the methods
for fabrication of GWS improve.
Thus one can ex-
This study was supported in part by Verein
Deutscher Ingenieure grant GILCULT 13N7963.
Soria acknowledges funding from the Generalitat de
Catalunya and the Ministerio de Ciencia y Tecnologia
(Spain) through the Ramon y Cajal program.
thank H. G. Weber and J. Zyss and their groups
for helpful discussions on GWS.
address is firstname.lastname@example.org.
S. Soria’s e-mail
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