1. AW1L.2.pdf CLEO:2014 © 2014 OSA
Photophysical properties of novel Ru-complex
probes for two-photon dissolved oxygen imaging
Aamir A. Khan*, Tahsin Ahmed, Genevieve D. Vigil, Susan K. Fullerton-Shirey, and
Scott S. Howard
Department of Electrical Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
akhan3@nd.edu
Abstract: Oxygen-sensitive hydrophobic indicators encapsulated in poloxamer nanomicelles
are quantitatively demonstrated to preserve the oxygen-sensitivity and the two-photon induced
phosphorescence in a ruthenium-complex indicator, thus providing for economical dissolved
oxygen imaging probes.
© 2014 Optical Society of America
OCIS codes: (180.4315) Nonlinear microscopy, (170.3650) Lifetime-based sensing.
1. Introduction
Quantitative measurement of dissolved oxygen is of crucial importance to many areas of industry and medicine [1].
Optical approaches for oxygen imaging are based on the principle of collisional quenching of a phosphorescence
indicator by oxygen molecules, which lowers both the average emission intensity (I) and emission lifetime (τ). This
phenomenon is described by the Stern-Volmer relationship, I0/I = τ0/τ = 1+Ksv · pO2, where (I0,τ0) are the emission
intensity and lifetime in the absence of oxygen, (I,τ) at a particular partial pressure of oxygen (pO2), and Ksv is the
Stern-Volmer constant. Lifetime measurements are preferred as they are self-referencing, insensitive to drift in most
experimental conditions, and form the basis of a powerful technique, fluorescence/phosphorescence lifetime imaging
microscopy (FLIM/PLIM), which when combined with multiphoton microscopy (MPM) [2], yields quantitative high-
resolution 3D images of oxygen concentration in the specimen.
Ruthenium(II) metal complexes are commercially accessible oxygen-sensitive indicators for a wide range of ap-
plications. [Ru(dpp)3]2+, a hydrophobic oxygen indicator, encapsulated in poloxamer nanomicelles [3, 4] has been
employed for qualitative oxygen imaging in aqueous media, both in vivo [5] and in vitro [3]. This paper demonstrates
and quantitatively characterizes that the encapsulated [Ru(dpp)3]2+ probes exhibit biologically important photophysi-
cal properties, such as lifetime sensitivity to dissolved oxygen and two-photon induced phosphorescence, thus making
them an attractive choice as an economical probe for two-photon imaging of dissolved oxygen.
2. Methods and Discussions
The encapsulated [Ru(dpp)3]2+ probes are prepared by separately dissolving [Ru(dpp)3]Cl2 in chloroform and polox-
amer 407 in water. The two solutions are mixed together and homogenized by ultrasonication, after which the chloro-
form is evaporated [3–5]. The absorption spectrum of the two samples, (a) ∼ 5 µM solution of [Ru(dpp)3]2+ in chlo-
roform and (b) ∼ 10 µM aqueous nanoemulsion of [Ru(dpp)3]2+ encapsulated in poloxamer, is measured by Perkin
Elmer Lambda 25 UV/VIS spectrometer. The emission spectra is similarly measured for both the samples through
Horiba Scientific Fluoromax-4 fluorometer by exciting at 425 nm. Fig. 1 shows that the results compare well for both
the samples.
Oxygen-sensitivity of the encapsulated [Ru(dpp)3]2+ probes is characterized by measuring the phosphorescence
lifetime (τ) as nitrogen and air are repeatedly diffused through the probe nanoemulsion (pO2 cycles between 0 and
213 hPa). The partial pressure of oxygen is measured by a commercial fiber-optic oxygen sensor, FireSting O2 (Py-
roScience GmbH, Germany). The nanoemulsion is excited via a pulsed UV-LED (365 nm) and the phosphorescence
is detected via Hamamatsu H7422PA-40 photomultiplier tube. SR400 photon counter (Stanford Research Systems,
CA) records the phosphorescence decay and measures the lifetime by rapid lifetime determination scheme [6]. The
(pO2,τ) measurements are synchronized in time and fitted to the Stern-Volmer equation. The resulting data, as shown
in Fig. 2(a), have a slightly concave curvature which is attributed to the nonuniform access of the oxygen molecules to
the core of the nanomicelle [7]. The data are still highly reproducible, nevertheless, and the calibration curves can be
generated for quantitative oxygen measurements.

2. AW1L.2.pdf CLEO:2014 © 2014 OSA
Two-photon absorption of the encapsulated [Ru(dpp)3]2+ probes is demonstrated by a custom-made two-photon
fluorospectrometer [3]. The two-photon induced phosphorescence intensity (F) is proportional to the average squared
intensity of light I(t)2 which in turn is proportional to the power of the excitation light (P), F ∝ I(t)2 ∝ P
[2]. The probes nanoemuslion is excited at 800 nm by Spectra Physics Mai Tai, a femtosecond Ti:S laser, and the
phosphorescence is measured by Hamamatsu H7422PA-40 photomultiplier tube. The result, as shown in Fig. 2(b),
demonstrates a pure two-photon induced phosphorescence in poloxamer encapsulated [Ru(dpp)3]2+.
350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Ru(dpp)3
2+
in chloroformRu(dpp)3
2+
in poloxamer
Absorbance(a.u.)
Emission(a.u.)
Figure 1. Comparison of absorption and emission spectra of [Ru(dpp)3]2+ when encapsulated in
poloxamer nanomicelles and when dissolved in chloroform.
0 30 60 90 120 150 180 210
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
τo
/τ
pO2
(hPa)
Measured Data
Least-Squares Fit
τ0 4.08 µs
Ksv 0.00847 hPa-1
RMSE 0.0515
τ0
τ = 1 + Ksv
· pO2
(a) (b)
Phosphorescenceintensity(a.u.)
10-1
100
101
Excitation power (mW)
Measured Data
Least-Squares Fit
y = A·x
2
Figure 2. (a) Stern-Volmer plot of the encapsulated [Ru(dpp)3]2+ probes. (b) Quadratically increas-
ing two-photon induced phosphorescence intensity against increasing excitation laser power in en-
capsulated [Ru(dpp)3]2+ probes at 800 nm.
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