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Multiphoton microscopy (MPM) allows for three-dimensional in vivo microscopy in scattering tissue with submicron resolution and high signal-to-noise ratio. MPM combined with fluorescence lifetime measurements further enables quantitative imaging of molecular concentrations, such as dissolved oxygen, with the same optical resolution as MPM, in vivo. However, biocompatible oxygen-sensitive MPM probes are not available commercially and are difficult to synthesize. Here we present a simple MPM oxygen imaging probe compatible with aqueous biological media based on a water-soluble ruthenium-complex nanomicelle. By adding a layer of silica shell to the nanomicelle assembly, oxygen sensitivity and probe stability in biological media increases dramatically. While uncoated probes are unusable in the presence of serum albumin, photophysical characterization shows that the silica coating enables quantitative oxygen measurements in biological media and increases probe stability by more than an order of magnitude.
© 2017 Optical Society of America
1. Introduction
Combining multiphoton microscopy (MPM) [1–3] and fluorescence lifetime imaging microscopy (FLIM) [4–6] enables three-dimensional quantitative microscopic imaging in living tissue. MPM-FLIM systems use the fluorescence lifetime of intrinsic molecular species to enhance contrast in label-free imaging of endogenous structures of living tissue [7–10] as well as obtain quantitative chemical measurements with fluorescent molecular probes [11–17]. Specifically, the quantitative imaging of dissolved oxygen in blood is of specific interest to many researchers in the biomedical community [18]. Oxygen is one of the most important molecules to biological processes and molecular imaging of oxygen at the tissue and cellular level plays a critical role in physiological research [12,14,15,19–25].
Quantitative MPM-FLIM measurement of oxygen is achieved by measuring the emission lifetime of oxygen-sensitive phosphorescent probes. The emission lifetime depends upon the rate of collision between dissolved triplet-oxygen and excited phosphorescent probe molecules, which in turn is a function of oxygen concentration and temperature. Probes based on platinum and palladium porphyrins such as PtP-C343 [22,26] and the Oxyphor family (R2, G2, R4, and G4) [27,28] produce highly sensitive measurements of dissolved oxygen in tissue and have been used for applications such as deep brain tissue and intravital imaging [11, 13, 14], blood flow monitoring [15], occlusion [12], and injury models [16]. Although oxygen-sensitive MPM-FLIM is an extremely powerful quantitative microscopy technique, oxygen-sensitive MPM-FLIM probes are not available commercially and are difficult to synthesize, thus limiting proliferation.
Recently, we have developed a simple technique to produce oxygen-sensitive MPM-FLIM probes by encapsulating a commercially available oxygen-sensitive ruthenium-complex ([Ru(dpp)3]2+) in a poloxamer surfactant (Pluronic F127) [29]. This approach allowed water-insoluble [Ru(dpp)3]2+ to form nanomicelle suspension in aqueous solutions with a relatively large two-photon optical response and near-optimal oxygen sensitivity. These [Ru(dpp)3]2+–nanomicelles are appropriate for MPM imaging and have been used to image mouse brain vasculature, in vivo [30,31].
While useful as a quantitative oxygen imaging probe in aqueous solutions, [Ru(dpp)3]2+–nanomicelles could not be used to perform reliable measurements of oxygen in biological media for two reasons. First, blood plasma contains proteins that bind to nanoparticles in order to facilitate transport (e.g., serum albumin). Bound proteins change the nanomicelle structure, reducing oxygen sensitivity [27, 28]. Second, nanomicelle stability is sensitive to dissolved ion concentration as the nanomicelles themselves are held together by steric stabilization. In saline solutions (e.g., blood), excess ions lead to demicellization. Demicellization is evident in [29] as the stable half-life of [Ru(dpp)3]2+–nanomicelles in phosphate buffered saline (PBS) is significantly lower than that in water. We now present a new approach to improve [Ru(dpp)3]2+–nanomicelle oxygen sensitivity and stability in biological media.
In this new approach, [Ru(dpp)3]2+–nanomicelles are coated in silica that enables collisional quenching of encapsulated [Ru(dpp)3]2+ in biological media and also acts as a binder to enhance micelle stability in the presence of electrolytes. An illustration of the probe is provided in Fig. 1. The method is adapted from the synthesis of an ordered mesoporous silica (OMS) material, COK-19 [32]. Silica is polycondensated within the micelle’s exterior polyethylene oxide (PEO) chains by adding sodium silicate to a buffered acidic micellar solution. This reaction provides mechanical scaffolding to the micelle by depositing a layer of silica on the micelle periphery that impedes PEO chain mobility. Experimental characterization of the silica coated micelles demonstrates that silica shell thickness is a function of the the amount of sodium silicate added to the buffered micelle solution [32]. While COK-19 requires micelle aggregation [32], our approach avoids aggregation in order to maintain the [Ru(dpp)3]2+–nanomicelle suspension in biological media by limiting the amount of silica deposition within micelles.
2. Materials and methods
Preparation of silica-coated [Ru(dpp)3]2+–nanomicelle consists of three main steps. First, [Ru(dpp)3]2+–nanomicelles are formed similarly as described in [29]. Second, silica is added to the outer polymer (PEO) chains of the micelles similar to that described in [32]. Finally, the resulting silica-coated [Ru(dpp)3]2+–nanomicelles are washed and suspended in PBS for use in biological media. The preparation procedure is depicted in Fig. 2 and described in detail below.
2.1. Precursors
Four precursors are required in this protocol: (1) surfactant, (2) dye, (3) acidic buffer, and (4) silica, as summarized in Table 1.
Table 1. List of precursors
484 mg of Pluronic F127 is added to 20 g of DI water in a glass vial and stirred for 30 minutes till completely dissolved. In a separate small vial, 200 μL of chloroform is added to 8 mg of [Ru(dpp)3]Cl2 dye and vortexed for 30 seconds. A 10× concentrated acidic buffer (pH = 3) is prepared by adding 1.715 g of citric acid monohydrate and 1.180 g of trisodium citrate dihydrate in 5 mL of DI water and stirred for 30 minutes until completely dissolved. Sodium silicate is purchased as a 35% w/w stock solution (Sigma-Aldrich) and reduced to a concentration of 0.9% w/w by diluting 3.5 g of stock solution in 10 g of DI water.
2.2. Nanomicelles preparation
10 mL of surfactant solution is poured in a 50 mL centrifuge tube. 100 μL of [Ru(dpp)3]2+ dye solution is gently pipetted deep in the liquid column to ensure complete transfer while avoiding splatters on tube walls. The tube is placed in an ice bath and sonicated with an ultrasonic homogenizer (Branson Sonifier SLPe 150 Watt). Sonication is performed for 10 s at 50% amplitude strength and is repeated 12 times with 10 s pauses in between. After the sixth sonication period, the tube is removed from the ice bath and vortexed for 30 s. The tube is then returned to the ice bath and the final 6 sonication periods are completed. After the sonication, the tube is placed in a 67 °C water bath for 30 minutes to evaporate the chloroform. The contents of the tube are filtered through a filter paper (Whatman grade 5) and the solution of suspended [Ru(dpp)3]2+–nanomicelles is collected.
2.3. Nanomicelles silica coating
Throughout this paper, silica coating level is described as percentage normalized to the amount of silica present in COK-19, an ordered mesoporous silica material with a similar preparation procedure [32]. This normalization was chosen to maintain consistency with existing literature.
The [Ru(dpp)3]2+–nanomicelle coating procedure is adapted from the COK-19 preparation procedure [32]. 1.8 mL of the [Ru(dpp)3]2+–nanomicelle solution is pipetted into a small glass vial and 0.2 mL of 10× buffer is added. While stirring vigorously with a magnetic stirring plate, S × 0.67 mL of diluted sodium silicate solution is carefully pipetted into the vial, where “S” is the desired silica coating level (e.g., “10%” requires 0.1 × 0.67 mL = 0.067 mL). Silica nucleation occurs instantaneously as sodium silicate is added to the buffered micelle solution, therefore vigorous stirring is crucial for uniform mixing and to avoid precipitation. The solution is stirred for 2 minutes to ensure completion. If a precipitate is formed or the solution turns turbid at this point, silica deposition has failed and the coating steps must be started over.
For characterization studies, micelle solutions are prepared at each of 0% (no coating), 10%, 20%, 30%, 40%, 50%, 60%, and 70% silica coating level. As shown previously [32] and confirmed here, the addition of sodium silicate corresponding to 70% silica coating level results in a turbid solution due to micelle aggregation, hence the usable range of silica coating level is up to ∼ 60%.
2.4. Serial washing with PBS
2 mL of silica-coated [Ru(dpp)3]2+–nanomicelles are poured in a Millipore Ultra-15 centrifugal filter unit (10 kDa MWCO; Sigma Aldrich). The tube is centrifuged at 5000 × g for 30 minutes at full acceleration/deceleration. Transparent filtrate indicates success; orange-tinted filtrate indicates nanomicelle failure as precipitated [Ru(dpp)3]Cl2 is present. The filtrate is discarded and all of the retained orange-tinted nanomicelle solution from the centrifugal filter tube is collected in a small glass vial. 0.5 mL of the collected nanomicelle solution is pipetted back into the original centrifugal filter tube. The solution is diluted 10× by adding 4.5 mL of PBS to the filter tube. The tube is centrifuged again with the same settings, the filtrate is discarded, and the retained solution is collected in a new vial. 0.5 mL of the new vial contents are pipetted back into the same centrifugal filter tube and diluted 10× by adding 4.5 mL of PBS. The tube is centrifuged for the third time and the retained liquid is collected as the final silica-coated [Ru(dpp)3]2+–nanomicelle product.
2.5. Phosphorescence lifetime measurement setup
Fluorescence or phosphorescence lifetime can be measured by either time-domain (TD) or frequency-domain (FD) methods [4]. Silica-coated [Ru(dpp)3]2+–nanomicelle probes are characterized using the FD scheme, which is convenient to implement while achieving near-optimal photon economy (i.e., signal-to-noise ratio) [33]. Phosphorescence lifetime measurements are obtained using a custom-made two-photon frequency-domain phosphorescence lifetime imaging microscope (MPM-FD-PLIM), outlined in Fig. 3. The excitation source is a Ti:S pulsed laser (Spectra Physics Mai Tai BB) with a repetition rate of 80 MHz and a pulse width of 100 fs. The laser is tuned to λ = 800 nm and the beam is modulated using an electro-optic modulator (EOM; Thorlabs EO-AM-NR-C1) driven by a 40 kHz sinusoidal signal. The power of excitation laser beam is controlled by a neutral density filter wheel to avoid phosphorescence saturation and is monitored by an optical power meter (Thorlabs S120C). The probe sample is held in a temperature-controlled 1 cm square glass cuvette. A commercial oxygen sensor (Pyro Science FireSting O2) is immersed in the sample to measure the dissolved oxygen concentration.
Fig. 3 Experimental setup for frequency-domain lifetime measurement with two-photon excitation. (EOM: electro-optic modulator. DAQ: data acquisition card. PMT: photomultiplier tube.)
Upon sinusoidal excitation, the probe sample produces a sinusoidal two-photon excited phosphorescence signal that is time-delayed relative to the excitation signal. Phosphorescence lifetime can be determined in the frequency-domain by using the relation, ωτ = tan ϕ, where ω, τ, and ϕ are the modulation frequency, phosphorescence lifetime, and phase difference between the excitation and the emission light signals respectively. A photomultiplier tube (PMT; Hamamatsu H7422PA-40) is used to measure the phosphorescence light signal from the sample. The analog electrical signal from the PMT is digitized and fed into a computer by a data acquisition card (National Instruments PCI-6110). The modulated excitation light signal from the optical power meter is also acquired and used as a reference channel for measuring the phase difference.
A computer program, written in MATLAB, coordinates triggering and data acquisition operations among several instruments that constitute the custom-made MPM-FD-PLIM system. The acquired data consist of 4 channels: (1) excitation signal, (2) emission signal, (3) partial pressure of oxygen, and (4) temperature. Fast Fourier transform is applied to extract the phase information of the fundamental frequency component of both the excitation and emission signals. Any phase shift in the emission signal due to optical and electrical path delays is accounted for by first calibrating the system using a fluorescence dye. The fast lifetime (< 10 ns) of the fluorescence dye calibrates away all the delays inherent in the optical and electronic components. Rhodamine B (τ = 1.7 ns) is used in these experiments as a calibration sample.
2.6. Phosphorescence lifetime measurements in blood mimic
Dissolved oxygen sensitivity of silica-coated [Ru(dpp)3]2+–nanomicelles in a blood mimic medium (2% albumin/PBS) is characterized using the lifetime measurement system described above. An oxygen scavenging enzymatic system consisting of glucose, glucose oxidase, and catalase is used to decrease the dissolved oxygen concentration in the probe sample as a function of time [22,26,34]. As shown in Fig. 3, the commercial oxygen sensor records the changes in pO2 while the lifetime measurement system records the τ values at each point in time. The two dataset are plotted against each other to generate the Stern-Volmer graph. Probe samples are prepared by diluting 20 μL of silica-coated [Ru(dpp)3]2+–nanomicelles with 2 mL of pre-aerated solution of blood mimic in a glass cuvette held at 37 °C. pO2 and τ data are continuously acquired while the oxygen scavenging enzymatic system is added to deoxygenate the sample. Data acquisition is stopped once the pO2 and τ both reach their asymptotic values.
3. Results and discussion
3.1. Probe formation mechanism
Pluronic F127 form spherical micelles in aqueous solution with a hydrophobic PPO core, sterically stabilized by a diffuse shell of hydrophilic PEO chains [35]. Pluronic F127 nanomicelles have been shown to form an outer silica shell (coating) when sodium silicate is added to micellar solution in an acidic buffer; the mechanism of this silica-coating formation is studied and characterized extensively by means of scanning tunneling electron microscopy (STEM), dynamic light scattering (DLS), and small angle X-ray scattering (SAXS) in [32]. The formation of spherical silica-coated micelles was confirmed through STEM examination; DLS studies show that nanomicelles hydrodynamic radius increases from 6.6 nm to 13.2 nm as the sodium silicate level is increased from 0 to 60%; and SAXS data further reveals that the addition of sodium silicate creates a silica shell of 1 nm to 4 nm thickness, as sodium silicate level increases from 10% to 60% [32].
3.2. Probe oxygen sensitivity
As the target application of this probe is intravital quantitative oxygen imaging in vivo, the probe is characterized for oxygen sensitivity in a medium that closely mimics mouse blood. Mouse blood plasma has a pH of 7.4 and contains albumin as the most abundant protein [36]. Albumin presents lipophilic binding sites to external agents in the blood, and is therefore well suited to evaluate oxygen sensitivity in blood [26–28]. To mimic both of the pH and lipophilic binding characteristics of the mouse blood, a 2% w/v solution of bovine serum albumin is prepared in PBS.
Oxygen sensitivity is described using the Stern-Volmer equation which relates the luminescence lifetime of a probe to oxygen concentration in its microenvironment. The relationship is given as,
where τ and τ0 are the probe’s lifetimes at a given partial pressure of oxygen (pO2) and at pO2 = 0 respectively. Ksv is the Stern-Volmer constant which determines the probe’s sensitivity to partial pressure of oxygen. Ksv is also the slope of the straight line when Stern-Volmer equation is plotted as given in Eq. 1; a steeper line signifies a higher oxygen sensitivity.The characterization is performed for seven probe samples with 0 − 60% level of silica coating, as defined in Sec. 2.3. It is observed that the probe with 0% coating (no silica coating) failed to produce valid lifetime measurement in the blood mimic medium. This occurs due to the incompatibility of the uncoated probe with biological media rich in lipophilic binding sites (e.g., serum albumin), thus making it impossible to use the uncoated nanomicelle probe for intravital quantitative imaging of oxygen in vivo.
The Stern-Volmer plots for the silica-coated [Ru(dpp)3]2+–nanomicelles probes in blood mimic medium at physiological temperature (37 °C) are shown in Fig. 4. It is evident from the plots that the coated probes show lower oxygen sensitivities in blood mimic (∼ 2 × 10−3 hPa−1) than that of the uncoated probe in water (8.47 × 10−3 hPa−1) [29]. The reason for the low sensitivity can be attributed to the added barrier to oxygen diffusion due to the silica shell deposited on the micelle assembly.
Fig. 4 Stern-Volmer plots for probes with different levels of silica coating at 37 °C. Inset: Variation of oxygen sensitivity with silica coating. Error bars represent standard deviation over 3 prepared batches. (1 hPa = 10−2 Pa = 1 millibar)
Fig. 4 also shows that increasing silica coating increases the coated probe’s oxygen sensitivity. This can be understood as thicker silica shell reduces the effects of albumin binding to the micelle. The additional protection offered by the silica coating increases the oxygen sensitivity by a factor of ∼ 2 as the silica coating level is increased from 10% to 50%, as seen in Fig. 4(inset).
In order to correctly determine the absolute value of oxygen partial pressure (pO2) from measured lifetime (τ), accurate values of the two Stern-Volmer parameters (Ksv and τ0) are required. However, both of these parameters are temperature dependent [37] and will invariably also be affected by the variations in the preparation procedure. Therefore, the values of these parameters must be determined for each batch of prepared probes by performing a simple two-point calibration (deoxygenated and air-saturated conditions) at the relevant temperature.
Table 2 lists the two Stern-Volmer parameters (Ksv and τ0) for different levels of silica coating. The variations around mean values are given as standard deviations over 3 prepared batches for each level of silica coating. Even though the variations are small, two-point calibration is still necessary and is a common practice with quantitative oxygen imaging probes to obtain high-confidence quantitative measurements [11–15].
Table 2. Stern-Volmer parameters for various levels of silica coating at 37 °C.
3.3. Probe bio-stability
Since probe stability is a strong function of dissolved ion concentration, uncoated nanomicelle stability is greatly reduced in PBS (half-life of 5 hours), compared to pure water and albumin in water samples (half-life on the order of days) [29]. Long-term stability characterization are therefore performed for various coated probes in PBS by observing the percent of micelles remaining in PBS solution as a function of time. Average emission intensity is used as a proxy for the intact micelle population in the sample, since the aggregated or demicellized [Ru(dpp)3]2+ dye molecules do not luminesce as [Ru(dpp)3]Cl2 is insoluble in water.
Fig. 5 shows nanomicelle probe stability in PBS as a function of silica coating level. As expected, an increase in silica coating increases the probe stability.
To gain more insight into the mechanism that improves stability, we model the nanomicelle decay profiles in Fig. 5 as a biexponential decay process:
where P(t) is the percent of the original micelles currently in solution at time t, hA and hB are the half-lives of the two processes, and A and B represent weighting coefficients attributed to the relative contributions of the two decay processes. The fraction of nanomicelles decaying with half-life hA can thus be estimated as A/(A + B). A biexponential model extremely accurately describes the experimental data (R2 > 0.999 for each silica coating level); representative fits are given in Figs. 6(a) and 6(b) for the 10% and 50% silica-coated probes, respectively. From these fits, the half-lives and the relative weights of the “slow” and “fast” processes can be found as a function of silica coating level. This analysis is presented in Fig. 6(c) and yields important insight into demicellization dynamics.Fig. 6 Two-decay micelle failure model. Decay profile of (a) 10%- and (b) 50%-coated probes and their biexponential fits. (c) Relative contributions and half-lives of the slow and fast decay process versus silica coating level.
Fig. 6(c) shows the existence of a fast decay process with a half-life of ∼0–2 hours, independent of silica coating level. At low silica coating levels (10%), approximately 60% of nanomicelles are limited by the fast process. However, the percent of nanomicelles limited by the fast decay is near zero for coating levels ≥ 30%, in which case nearly all nanomicelles will exhibit the slow demicellization process. The half-life of the slow process increases with increasing silica coating level until a critical silica concentration is reached, after which micelles aggregate into ordered structures [32] and half-life decreases (e.g., silica coating of ≥ 60% in Fig. 6(c)).
We attribute the fast decay process to the population of nanomicelles which remain “uncoated” (i.e., do not experience silica nucleation). The demicellization rate of uncoated nanomicelles (i.e., fast half-life) is independent of the amount of sodium silicate added. However, the probability of a nanomicelle remaining uncoated decreases with increasing levels of sodium silicate. On the other hand, the slow process is attributed to the loss of nanomicelle electrostatic equilibrium in the presence of dissolved ions. When sodium silicate is added to the emulsion, it preferentially nucleates a condensation reaction within the PEO chains of the surfactant, leading to a silica shell around the dye–poly(propylene oxide) (PPO) core. The silica acts as a scaffold that mechanically supports the micelle by diminishing PEO chain mobility [32], making it less sensitive to electrostatic imbalances caused by ions in the solution. As a result, the half-lives of silica-reinforced nanoprobes increase with increasing silica concentration until the critical aggregation level is reached.
3.4. Summary
This paper presents a simple method to prepare silica-coated [Ru(dpp)3]2+–nanomicelle probes for quantitative MPM-FLIM oxygen imaging. The new probe significantly improves both the oxygen sensitivity and the bio-stability compared to previous work [29]. The results are summarized in Fig. 7 and show that both the oxygen-sensitivity and the long-term stability increase with increasing silica coating. Therefore, the amount of silica should be chosen to be as high as possible prior to the the nanomicelle aggregation point (silica coating level ≈ 50%).
The silica-coated [Ru(dpp)3]2+–nanomicelle probe described here enables two-photon quantitative oxygen imaging in vivo using a commercially available, inexpensive dye such as [Ru(dpp)3]Cl2 and off-the-shelf reagents. The probe offers high oxygen sensitivity (2.4 × 10−3 hPa−1) and a very long bio-stable life (∼80 hours). Coupled with the ease of preparation, this probe can be readily deployed to promising applications such as 3D, high-resolution oxygen imaging of deep brain blood vessels in vivo.
Funding
This material is based upon work supported by the National Science Foundation under Grant No. CBET-1554516.
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