1) Ruthenium-poloxamer nanoprobes were prepared by encapsulating the hydrophobic ruthenium dye [Ru(dpp)3]Cl2 in poloxamer 407 surfactant micelles, providing a simple method for aqueous solubilization. 2) Two-photon microscopy of a mouse brain in vivo was performed using the probes, demonstrating excellent imaging quality and contrast of the brain vasculature down to 100 μm resolution. 3) The probes showed good two-photon excitation properties, oxygen sensitivity through lifetime measurements, and stability, indicating potential for quantitative three-dimensional oxygen concentration mapping in tissues.

1. BT1A.3.pdf Optics in the Life Sciences 2015 © OSA 2015
Two-Photon Intravital Imaging of a Mouse Brain
In Vivo Using Easily Prepared
Ruthenium-Poloxamer Nanoprobes
Aamir A. Khan,1∗ Siyuan Zhang,2,3 Genevieve D. Vigil,1
Joel D. Boerckel,4 and Scott S. Howard1
1Department of Electrical Engineering, 2Harper Cancer Research Institute, 3Department of Biological Sciences, and
4Department of Aerospace and Mechanical Engineering. University of Notre Dame, Notre Dame, IN 46556, USA.
*E-mail: akhan3@nd.edu
Abstract: A hydrophobic, oxygen-sensitive ruthenium-complex dye is prepared in water
through a simple surfactant micellization technique. Two-photon imaging of a mouse brain
vasculature in vivo is demonstrated using the probe with excellent detail and contrast.
© 2015 Optical Society of America
OCIS codes: (180.4315) Nonlinear microscopy, (170.1460) Blood gas monitoring.
1. Introduction
High-resolution, in vivo imaging of brain has garnered a lot of interest lately among the imaging optics, neurophysics,
and neurophysiology researchers alike [1,2]. Traditional techniques like confocal fluorescence microscopy use ultra-
violet or visible light for excitation which causes high scattering in the living tissue and results in low penetration and
poor signal-to-noise ratio. These problems can be avoided by utilizing advanced nonlinear optical methods such as
two-photon microscopy. The benefits of nonlinear optics based imaging modalities stem from the two facts: (a) the use
of near infrared light for excitation, which scatters significantly less in the living tissue, provides for deeper penetration
and higher signal-to-noise ratio and (b) two-photon induced luminescence is extremely localized which enables 3D
imaging without requiring a pinhole and provides for a much better photon economy.
While advances in two-photon microscopy technology allow for faster and deeper imaging in vivo, quantitative
chemical imaging still remains challenging. There exists only a few probes that show sensitivity to biologically relevant
chemical species such as dissolved oxygen [3]. An even fewer of them are water-soluble which makes high-resolution
quantitative oxygen imaging in vivo difficult and expensive. In the search of hydrophilic dyes that are sensitive to
dissolved oxygen, several techniques have been developed, such as attaching hydrophilic dendrimers and immobilizing
the dye in a polymer matrix [4].
Our work presents another technique to prepare hydrophobic dyes for aqueous media applications. We achieve
this by encapsulating an oxygen-sensitive hydrophobic dye, [Ru(dpp)3]Cl2,† in a surfactant, poloxamer 407. The
paper describes the preparation method and the photophysical properties of the probes in Sec. 2 and 3 respectively.
3D intravital imaging of a mouse brain in vivo using ruthenium-poloxamer probes with a commercial multiphoton
microscopy setup is shown in Sec. 4. The last section (Sec. 5) discusses the future work with the nanoprobes and some
potential applications.
2. Preparation of ruthenium-poloxamer nanoprobes
The problem with most of the methods for solubilizing hydrophobic dyes in water is that they rely on expensive and
complex chemical synthesis procedures. In contrast, our proposed method does not require any chemical synthesis and
is therefore relatively simple and quick [4]. The method utilizes a surfactant micellization technique and is adapted
from a procedure reported by Maurin et al. for preparing a hydrophobic dye for intravital imaging in vivo [5,6]. Other
groups have utilized similar techniques to use hydrophobic dyes for intracellular oxygen imaging [7]. In our method,
we use poloxamer 407 surfactant which is a non-ionic block copolymer of the form PEO–PPO–PEO, where PPO
is polypropylene oxide and the PEO is polyethylene oxide. Because PEO is hydrophilic and PPO is hydrophobic,
†Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride.

2. BT1A.3.pdf Optics in the Life Sciences 2015 © OSA 2015
700 800 900 1000
10
15
20
25
30
35
40
Excitation wavelength (nm)
σ
TPE
(GM)
760 nm
1.5 2 2.5 3 3.5 4
0
50
100
150
200
τ (µs)
pO
2
(hPa)
pO
2
(τ) = 1620 e
−1.68τ
+ 257 e
−0.312τ
− 72.3
(a) (b)
Figure 1. (a) Two-photon excitation cross-section spectrum and (b) lifetime versus oxygen partial
pressure calibration curve of ruthenium-poloxamer nanoprobes.
micelles are formed upon emulsification with the hydrophobic dye sequestered in the center with PPO blocks and the
hydrophilic PEO blocks on the periphery.
The proposed method is very simple and takes less than two hours to prepare [Ru(dpp)3]2+ encapsulated in polox-
amer 407. We begin by dissolving 4 mg of [Ru(dpp)3]Cl2 dye in 100 µL of chloroform and separately dissolving
100 mg of poloxamer 407 in 100 mL of water. The two solutions are thoroughly mixed in a 50 mL centrifuge tube and
then emulsified using a high energy ultrasonic stirrer (Branson Sonifier® SLPe 150 Watt). Chloroform is evaporated
by keeping the emulsion in a 62 ◦C water bath for 30 minutes while the excess surfactant is removed by spinning the
emulsion at 5000 g for 30 minutes in a 10 kDa molecular weight cut-off centrifugal filter (Amicon Ultra-15; EMD
Millipore). The resulting concentrate is the ruthenium-poloxamer nanoprobe solution and contains only the suitably
sized micelles (> 5 nm) that do not cross the blood brain barrier. The concentration of [Ru(dpp)3]2+ in the nanoprobe
solution is measured to be 1400±128 µM.
3. Photophysical properties
Ruthenium-poloxamer nanoprobes show linear (one-photon) absorption and emission peaks at 442 nm and 629 nm
respectively with an emission quantum yield of 0.3. The two-photon excitation cross-section (σTPE) of the probes is
also measured and is shown in Fig. 1a. The cross-section peaks at 760 nm with a value of 37.3 ± 11.7 GM [4]. In
comparison, the σTPE-max values for fluorescein and PtP-C343 (a well-established oxygen-sensitive probe for intravital
imaging) are 34±8.7 GM and 13 GM respectively [3,8]. The large value of σTPE-max makes the ruthenium-poloxamer
probes a viable alternative for two-photon imaging as shown in the next section.
Oxygen-sensitivity of the probes is also characterized by measuring the phosphorescence lifetime (τ) as a function
of dissolved oxygen concentration (partial pressure of oxygen; pO2). The emission decays faster (lifetime drops) as
more oxygen molecules (large pO2) quench the phosphorescing dye molecules through a non-radiative decay process
called collisional quenching. As shown in Fig. 1b, a calibration curve is generated from the results of the charac-
terization experiments and is fitted to an empirical model, pO2(τ) = A1e−α1τ +A2e−α2τ/ + p0. The results show a
large dynamic range of lifetime (1.5–4.2 µs) for oxygen levels ranging from air-saturated to fully hypoxic conditions,
indicating that [Ru(dpp)3]2+ dye retains sensitivity to dissolved oxygen after encapsulation in poloxamer micelles [4].
The nanoprobes are visually observed to remain stable in DI water at room temperature for more than 6 months.
Stability of the probes is also tested quantitatively in several biological media such as bovine serum albumin (BSA),
phosphate buffered saline (PBS), mouse blood, cell culture growth medium, and different combinations of the above
species. It is found that the probes demicellize most rapidly in PBS solution with a half-life of 5 hours, thereby readily
surpassing the time required for in vivo imaging experiments.
4. Intravital imaging
To demonstrate the two-photon imaging capabilities of ruthenium-poloxamer nanoprobes, intravital imaging on a
mouse brain in vivo is performed. A healthy lab mouse (FVB female) is chosen for the study. While the mouse is kept
under general anesthesia by means of isoflurane inhalation, a cranial window is opened in order to get optical access

3. BT1A.3.pdf Optics in the Life Sciences 2015 © OSA 2015
100 µm 100 µm
0 µm
100 µm
200 µm
(b)(a)
Figure 2. Perspective intensity projection and 3D reconstruction of two-photon imaging of a mouse
cortex vasculature in vivo.
to the cortex vasculature.‡ The mouse is allowed to recover from the traumatic effects of surgery for approximately 4
hours prior to imaging. Again inducing general anesthesia through isoflurane, 100 µL of ruthenium-poloxamer probe
solution (1400µM) is retro-orbitally injected into the blood stream. The mouse is placed on the stage of a commercial
multiphoton laser scanning microscope (Olympus Fluoview FV1000MPE) for 3D imaging of the cortex vasculature.
Two-photon excitation of the nanoprobes in the bloodstream is achieved by focusing a 820 nm beam from a mode-
locked Ti:S laser (Spectra Physics Mai Tai DeepSee; 80 MHz rep. rate; 100 fs pulse width) through a water immersion
objective lens (Olympus XLPLN-25X-WMP-1.05NA). 100 XY-scans are acquired at a depth increment of 2 µm,
where each XY-scan is composed of 640×640 pixels at a resolution of 0.795 µm/pixel with a dwell time of 10 µs/pixel.
The emission is detected in a passband of 650–700 nm around the emission peak of [Ru(dpp)3]2+. The results of
imaging are presented as perspective intensity projection along the Z-axis and as 3D reconstruction of the acquired
area of the cortex in Fig. 2a&b respectively. The results show excellent detail and contrast thereby demonstrating the
viability of using [Ru(dpp)3]2+ encapsulated in poloxamer as simple and inexpensive probes for two-photon intravital
imaging in vivo.
5. Conclusions
Ruthenium-poloxamer nanoprobes have amply demonstrated excellent two-photon imaging capability and good sen-
sitivity to dissolved oxygen. Though the imaging results shown here are only quantitative (intensity based), the probes
can readily be used in a lifetime imaging system to yield 3D, high-resolution, and quantitative oxygen concentration
maps in blood, cytosol, and other biological and aqueous media. These imaging systems can have potential applica-
tions in blood oxygen monitoring, cellular activity imaging, and high-resolution brain imaging, etc.
References
1. K. Wang, N. G. Horton, and C. Xu, Opt. Photon. News, 24, 32–39 (2013).
2. S. Sakadˇzi´c, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor,
E. H. Lo, S. A. Vinogradov, and D. A. Boas, Nat. Methods, 7, 755–759 (2010).
3. A. Y. Lebedev, T. Troxler, and S. A. Vinogradov, J. Porphyrins Phthalocyanines, 12, 1261–1269 (2008).
4. A. A. Khan, S. K. Fullerton-Shirey, and S. S. Howard, RSC Adv., 5, 291–300 (2015).
5. M. Maurin, O. St´ephan, J.-C. Vial, S. R. Marder, and B. van der Sanden, J. Biomed. Opt., 16, 036,001 (2011).
6. S. S. Howard, A. Straub, N. G. Horton, D. Kobat, and C. Xu, Nat. Photonics, 7, 33–37 (2013).
7. X.-d. Wang, J. a. Stolwijk, T. Lang, M. Sperber, R. J. Meier, J. Wegener, and O. S. Wolfbeis, J. Am. Chem. Soc.,
134, 17,011–17,014 (2012).
8. C. Xu and W. Webb, J. Opt. Soc. Am. B, 13, 481–491 (1996).
‡All lab animal procedures are approved and regulated by the University of Notre Dame IACUC.