Spatiotemporally controlled nano-sized third harmonic generation agents.

Here, we present a new class of third harmonic generation (THG) imaging probes that can be activated with precise spatiotemporal control using non-linear excitation. These probes consist of lipid-coated perfluorocarbon nanodroplets with embedded visible chromophores. The droplets undergo phase transition from liquid to gas upon heating mediated by two-photon absorption of NIR light by the embedded dyes. Resulting microbubbles provide a sharp, local refractive index mismatch, which makes an excellent source of THG signal. Potential applications of these probes include activatable THG agents for biological imaging and "on-demand" delivery of various compounds under THG monitoring.


Introduction
Advances in imaging technologies and respective contrast agents have greatly expanded our understanding of tissue structure, cell functions and interactions as well as subcellular events from basic cell biology to oncology [1][2][3][4][5]. Advanced microscopy exploits a range of contrast strategies to detect structural, molecular, and physical cell and tissue properties, and there is growing need for pairing of imaging modalities with innovative contrast agents.
Most contrast agents in life sciences today rely on the principle of single-or two-photon excitation. While these agents are extremely useful in generating imaging contrast, there are constraints in the number of unique targets that can be imaged due to spectral overlap in the visible light range. Additionally, scattering and absorption of both the visible excitation sources and emissions by the contrast agents present a challenge to 3D in vitro and in vivo imaging [6]. The development of multiphoton microscopy with pulsed near-red and infrared laser excitation sources enabled in vivo imaging of live cells and tissue structures at high resolution, low phototoxicity, and reasonable depth of penetration (300-1000 µm) [7][8][9][10][11]. Furthermore, the possibility of higher harmonic imaging by second harmonic generation (SHG) and third harmonic generation (THG) enables additional modes of endogenous tissue contrast and opportunities for development of exogenous contrast agents which report by fluorescence-independent mechanisms. SHG occurs when biological structures containing asymmetric repetitive units (e.g. striated muscle and collagen fibers) convert two photons of an excitation light source into a single emitted photon at double the energy (1/2 of the excitation wavelength). THG occurs at interphases, typically with a refractive index mismatch (e.g. water-lipid/protein), that results in conversion of three photons of an excitation light into a single emitted photon with triple the energy (1/3 of the excitation wavelength) [12]. Whereas SHG is limited to asymmetry of the structure being imaged, THG is based on differences in refractive index and, hence, is more broadly applicable. Unlike fluorescence, THG does not suffer from photobleaching or generation of reactive oxygen species [12] and allows detection of intrinsic complex tissue structures with subcellular resolution, including interstitial tissue, cell surfaces, and microvesicles in live animals [13].
Such advantages prompted development of specific THG imaging contrast agents for specialized applications in life sciences requiring unique labeling or increased contrast. For biologically relevant THG imaging, these agents include lipid-enclosed quantum dots [14], lipid microbubbles [15], tattoo dye [16], porphyrin aggregates [17], bismuth ferrite [18] or titanium dioxide [19] nanoparticles. These constitutive THG probes can enable cell tracking applications [14,18], as well as molecular imaging through conjugation with targeting moieties, e.g., antibodies or peptides [15]. However, they also suffer from the complexity of THG signals originating in 3D tissue and the generic nature of the signal which might not be readily discriminated from the background. To increase specificity, activatable or externally triggered THG probes could offer new sensing capabilities in biomolecular imaging by creating on demand contrast or enabling image-guided interventions and delivery, not unlike activatable fluorescence probes [20][21][22][23][24]. Here we address this need by developing and initially validating spatiotemporal, inducible contrast probes for THG imaging that are based on phase changing perfluorocarbon (PFC) nanodroplets. We applied highly localized twophoton excitation to trigger phase-change of PFC nanodroplets and THG visualization of the resulting microbubbles post-excitation dynamics. The spatiotemporal precision of nanodroplet activation could allow utilization of these agents for spatially and temporally controlled on-demand image-guided delivery, which is not possible with previously reported agents.
The nanodroplets consist of a lipid-stabilized PFC core with embedded light absorbing dye molecules. Previously, these nanoparticles were used to create a high signal and contrast in photoacoustic imaging [25][26][27]. In these studies, excitation of the nanodroplets occurs with nanosecond laser pulses, which are matched with the absorption band of an embedded dye, followed by a liquid to gas transition of the PFC core which elicits a strong photoacoustic signal. Using this principle, we recently demonstrated that the phase changing PFC nanodroplets can also be used to create localized, high frequency elastic waves in optical coherence elastography measurements [28,29]. PFC with relatively low boiling temperatures, e.g. perfluoropentane or perfluorohexane, are generally used to facilitate the liquid-gas phase transition. These compounds are highly inert and non-toxic materials and have been successfully used in variety of biomedical applications including blood substitutes [30][31][32][33][34][35] and in vivo imaging [36,37].
We hypothesized that after conversion of a nanodroplet into a microbubble, the distinct interphase between aqueous surrounding and the PFC gas will provide a sharp, local refractive mismatch and therefore elicit THG. To demonstrate applicability of these nanodroplets in 3D settings, we applied infrared two-photon excitation of the embedded dye molecules to promote liquid to gas phase transition of PFC nanodroplets and recorded precise, spatiotemporally controlled nanodroplet activation. The results reveal a first-in-field phasechange nanodroplet system for spatially and temporally controlled and activatable probes for THG imaging.

Synthesis and assessment of nanodroplets
The nanodroplets were synthetized from a liquid dodecafluoropentane core coated by a mixture of phospholipids and Cyanine 3 (Cy3) dye molecules. The formulation was adopted from a previously described protocol [28]. Briefly, 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethyleneglycol-2000 (DSPE-PEG-2000), and cholesterol (all from Avanti Polar Lipids, Inc.) were dissolved in 2 mL of chloroform at a weight ratio of 90:8.5:1.5, respectively, and the total mass of 20 mg. The mixture was dried in a rotary evaporator (Cole-Palmer Instrument Company, LLC.) at 40°C for 30 minutes to form a lipid cake. Then, 2 mL of deionized water was added to the lipid cake and the vial was shaken at 250 rpm on ice for 30 minutes. Simultaneously with the lipid hydration process, 125 µL of dodecafluoropentane (FluoroMed L.P.) was mixed with 100 µL of 1% (v/v) aqueous solution of 1H,1H,2H-Perfluoro-1-hexene,3,3,4,4,5,5,6,6,6-nonafluoro-1-hexene (Zonyl PFBE, Sigma-Aldrich Corp.) and 150 µL of 2 mg/mL aqueous solution of Cy3 dye (Lumiprobe Corp.). This core mix was vortexed for 20 seconds and then sonicated in a benchtop ultrasonic bath CPX-962-218R (Fisher Scientific) in ice-cold water for 30 seconds. Subsequently, 2 mL of rehydrated phospholipids were added to the core mixture of PFC, Cy3 dye, and Zonyl PFBE. The suspension was vortexed for 30 seconds and then sonicated in the benchtop ultrasonic bath in ice-cold water for 1 minute. Final sonication was carried out using the VCX 500 ultrasound probe with a 2 mm tip (Cole Palmer) for two 1 minute cycles at 25% maximum amplitude; the cycles were separated by 20 seconds of vortexing. Then, the suspension was kept on ice for 5 minutes and was washed twice with water at 3100 G for 15 minutes to remove the excess of free dye and lipids. The final pellet of washed nanodroplets was re-suspended in 1 mL of deionized water. Blank nanodroplets (containing no dye) were synthesized using the same protocol except deionized water was added to the core mix instead of the dye solution.
Average size of the nanodroplets was measured by dynamic light scattering using intensity (Delsa Nano C, Beckman Coulter, Inc.). To confirm dye inclusion inside the nanodroplets, fluorescence intensity of blank and dye-containing preparations were measured with Synergy HT microplate reader (BioTek Instruments, Inc.) using 530/25 and 590/35 nm excitation/emission filters.
PFC concentration in nanodroplet preparations was measured using 19 F NMR. Briefly, 10 µL of a nanodroplet suspension were mixed with 400 µL of deionized water, and 75 µL of 0.5% reference solution of trifluoroacetic acid in deuterium oxide (both from Sigma-Aldrich Corp.) in a 5 mm NMR sample tube (Wilmad-LabGlass). One-dimensional 19 F NMR was performed with a 500 MHz NMR spectrometer (Bruker Corporation). Typical acquisition parameters included acquisition time of 0.577 s, signal averages -16 (with phase cycling) and a relaxation delay of 15 s. Spectral amplitudes were corrected for incomplete relaxation and fluorine quantification was carried out by integrating peaks and comparing the sample's peak to the reference peak from trifluoroacetic acid standard.

Phantom preparation
Polyacrylamide (10%, PAA) phantoms were made by adopting a previously described protocol [38]. Briefly, 125 µL of 40% acrylamide aqueous solution (Ambion Inc.), 7.5 µL of 438 mM aqueous solution of ammonium persulfate (Sigma-Aldrich Corp.), 0.75 mg of nanodroplets (in terms of PFC concentration, the corresponding volume was varied depending on a preparation but was typically 20-60 µL), 2 µL of tetramethyl ethylenediamine (Sigma-Aldrich Corp.), and deionized water to bring the final volume up to 500 µL were mixed together and poured inside of a 20 mm diameter and 2 mm high silicone isolator (Grace Bio-Labs) attached to a macroscopy slide. Isolators were sealed with a 24 x 24 mm cover glass (Fisher Scientific) and the mixture was allowed to polymerize to a 10% PAA gel at room temperature for 20 minutes before imaging.

Multiphoton microscopy imaging
Imaging was performed on a custom multiphoton microscope (TriMScope-II, LaVision BioTec), equipped with three tunable Ti:Sa (Coherent Ultra II Titanium:Sapphire) lasers and two Optical Parametric Oscillators (OPOs; Coherent APE). Power under the objective was controlled by attenuators in the beams paths and measured before each experiment. A longworking distance, 25x NA 1.05 water immersion objective (Olympus) was used for image acquisition. Multi-spectral detection was performed using up to 3 GaSP photomultipliers (PMTs, Hamamatsu) in the backward configuration using sequential single or dual excitation wavelengths scans for z-stack, zoom and overview, and time lapse acquisitions. The following excitation and emission channels were collected where indicated: Lifeact-GFP and calcein AM (920 nm; 525/50 nm); H2B-mcherry (1090 nm or 1100 nm; 620/60 nm), SHG (1090 nm; 525/50 nm), and THG (1280 nm; 420/50 nm). Nanodroplets were excited with 1090 nm or 1100 nm two-photon excitation (100-200 mW measured under the objective) and phase-change induced THG signals were detected using 1280 nm multi-photon excitation using 80-110 mW under objective in cell culture in vitro which is comparable for imaging in the mouse dermis in vivo. Time-lapse recordings were performed by acquiring images every 2 seconds for 200 to 400 seconds. The 1090 nm or 1100 nm wavelength for nanodroplet activation was applied for continuous intervals to generate THG signals. Single time point, 3D multichannel z-stacks were acquired with z step sizes of 3 µm. Scan fields had a pixel dwell time of 4 µs (imaging in ND suspensions) and 8.9 µs (imaging in gel phantoms and collagen matrix).

THG signal and image analysis
THG signal and image analysis was performed using Fiji/ImageJ2 [39], Excel (MS) and results were displayed with GraphPad Prism 7. To measure the dependence of signal generated by activated PFC nanodroplets on excitation power, Cy3 PFC nanodroplets were first excited using 1100 nm (~110 mW) in imaging media, phosphate buffered saline with Ca 2+ and Mg 2+ (Sigma-Aldrich Corp) and 10% fetal bovine serum (Cellgro) mixed with 20 µL of nanodroplets, and 1280 nm excitation (~90 mW) to obtain microbubble signal emission (420/50 nm). Once microbubbles were detected, photoactivation at 1090 nm was ceased and 1280 nm excitation over a range of available powers (89-186 m W) was used alone to collect single images from the induced microbubbles at a single focal plane. The mean intensity of 5 microbubbles from 4 independently excited positions in cell culture was quantified using MaxEntropy thresholding (Fiji). Signal-power data set were log-log transformed and individually fitted using a linear regression. The R 2 values for all 5 fits was > 0.97. The mean of the slopes (slope m ) determined from each data set was calculated and reported with the standard deviation (slope m = 2.92 ± 0.53). For display, all log-log transformed data sets were plotted together with a fit line (mean slope and y-intercept of the individually modeled data sets).
Quantification of THG signals induced at defined z positions ( Fig. 4(a)) in PAA gels was performed by first measuring the raw integrated density (total intensity) of each z slice of the acquired 3D stack. Background signal was estimated by using the Yen threshold method in Fiji and measuring the raw integrated density (bkg-intensity) for the identified background area for each z slice. Final THG intensity (AU) was reported as the difference between the total intensity and the bkg-intensity as determined above and then plotted as a function of the z position (μm).
Size distribution of THG microbubbles formed in solution was estimated from time-lapse imaging data. Background intensity of time-lapse recordings was estimated by measuring the mean intensity of the first 25 images (where no nanodroplets were excited). All time-lapse images were background corrected with this value. THG signal was discriminated from background using the Moments threshold method in combination with particle analysis to identify THG positive events of at least 2 square pixels in dimension (~0.6 µm 2 ). The areas of all identified THG pixels were measured. Assuming that all THG microbubbles are spherical upon formation and appear circular during single focal plane detection, the measured areas were converted into an 'apparent' circular diameter for size distribution analysis.

Cell culture, collagen matrix embedding and viability test
Mouse mammary 4T1 carcinoma cells stably expressing nuclear (H2B-mCherry) and actin filament binding (Lifeact-GFP) fluorescent reporters were maintained at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma-Aldrich Corp.) supplemented with 10% fetal bovine serum (Cellgro Corning), 100 U/mL penicillin and 100 µg/ml streptomycin (Hyclone ThermoFisher) and 1% sodium pyruvate (ThermoFisher). Cells were detached with EDTA (1mM) and trypsin (0.5%) and the collected cell suspensions were incorporated into type I collagen from rat-tail tendon (BD Biosciences; final concentration 2.5 mg/mL) followed by gel polymerization at room temperature as described previously [40,41]. Using either the same silicon isolator and setup as for the PAA phantoms or a 35 mm glass bottom µ-Dish (ibidi GmbH), polymerized cell-containing collagen matrix was overlaid with culture or imaging media (1-3 mL) mixed with 40-120 µL of PFC nanodroplets in water and imaged after 20-30 minutes.

Nanodroplets characterization
The nanodroplets consisted of a liquid PFC core coated with a mixture of PEGylated phospholipids, and embedded Cy3 dye molecules ( Fig. 1(a)). The phospholipid coating stabilizes PFC nanodroplets in an aqueous environment while PEG provides protection from nonspecific interactions and Cy3 chromophores with an absorbance maximum at 550 nm were embedded to enable two-photon absorption and photoactivation. Nanodropletcontaining aqueous solution displayed characteristic purple coloring when Cy3 dye was embedded ( Fig. 1(b)). Typical preparations resulted in fairly uniform size distributions of nanodroplets with average sizes at the major distribution peak of 379 ± 96 nm and 306 ± 80 nm for PFC nanodroplets with embedded Cy3 chromophores (ND-Cy3) and "blank" PFC nanodroplets without dye molecules (ND-B) specimen, respectively ( Fig. 1(c)). Fluorescence intensity measurements confirmed dye inclusion inside the ND-Cy3 nanodroplets ( Fig. 1(d)).

Two-photon activation of PFC phase-change nanodroplets
The nanodroplets were designed to enable an externally triggered phase-change by incorporating a visible dye to absorb incoming NIR photons through a two-photon absorption process. The choice of the perfluoropentane as the core material was driven by its relatively low boiling temperature of 29°C in bulk. It is important to note, that the boiling temperature is inversely proportional to nanodroplet size due to increase in the surface tension [42][43][44]. We hypothesized that two-photon excitation would induce a liquid-gas phase change of the PFC core through the highly localized heating created by the embedded chromophores resulting in an interface between aqueous surroundings and the PFC microbubble that can be detected by THG microscopy.
To confirm this hypothesis, we activated ND-Cy3 with 1100 nm two-photon laser excitation (or 1090 nm) tailored to the predicted two-photon absorption peak of Cy3. Previously, it was shown that two-photon fluorescence of Cy3 labeled IgG immunoglobulins can be excited in the range of ~930-1150 nm with the maximum two-photon fluorescence at 1032 nm [45]; this data indicate that the optimum two-photon absorption peak of Cy3 might differ from the predicted value. However, our two-photon imaging system did not have sufficient power at wavelengths below 1080 nm for nanodroplet activation, therefore, we used the 1100 nm excitation. Any THG signals resulting from the phase transition were excited using a higher infrared wavelength (1280 nm), which does not excite Cy3 dye, and detected at 395-445 nm, which is below the Cy3 emission.
Each nanodroplet preparation with or without the dye was embed in 10% PAA phantoms. All samples were continuously imaged (image acquired every 2 seconds) with a 1280 nm laser for the duration of the experiments (200 to 400 seconds) in order to detect any phasechange signals (Fig. 2(a) schematic). We first confirmed that continuous 1280 nm excitation alone over a period of 50 seconds did not elicit any signals in ND-Cy3 samples (Fig. 2(b)). Addition of the Cy3 excitation laser (1100 nm) resulted in formation of phase-change signals in the THG emission channel (Fig. 2(c)). After discontinuation of 1100 nm excitation, the phase-transition signal originating from microbubbles persisted for at least 50 sec (Fig. 2(d)). In contrast, little to no signal was induced when 1100 nm excitation was applied to dye-free ND-B blank nanodroplets for extended time periods (350 sec) (Figs. 2(e)-2(g)). A small number of sporadic phase-transition signals observed in some ND-B samples after a long observation period are most likely due to spontaneous phase change in some of the bigger and thus less stable PFC nanodroplets (Fig. 2(g), arrowhead). Fig. 2. Phase-change of nanodroplets with applied two-photon excitation. (a) Schematic of the experiment. Note, that both laser beams were delivered through the same objective. Single focal planes in gels with embedded ND-Cy3 (b, c, d) and ND-B (e, f, g) were continuously imaged with 1280 nm excitation for phase-change signal detection. No signal was registered in ND-Cy3 sample before 1100 nm excitation that was started after 50 seconds (b). Phase-change signal was registered during (c) 1100 nm laser excitation and (d) after its removal at 350 seconds. A similar sequence with extended 1100 nm excitation showed minimal signals for ND-B (e) before, and (f, g) during 1100 nm laser excitation starting after 50 and ending at 400 seconds; an arrowhead points to a rare, sporadic event. Scale bars = 10 µm.
In general, the liquid-gas phase transition is expected to result in up to ~5-6 fold volume increase based on the difference in density between gaseous and liquid phases of PFC [42,46]. However, previous studies of ultrasound activated PFC phase transition from nano-or microdroplets to microbubbles showed a volume increases in excess of 10-fold [42,[46][47][48][49]. The extra bubble expansion was shown to be associated with diffusion of dissolved gases from the surrounding media into the activated microbubbles due to an order of magnitude higher solubility of some gases in perfluorocarbon in comparison to water [50]. Consistently, PFC nanodroplets, with an average initial size just under 400 nm, reached sizes of ~4 µm in diameter afte occasionally a growth to di extensively st over a perio mechanisms w form larger se or gaseous P [49,53,54]. T movement of Overall, o photon absorp subsequent m the proof of p for multiphoto

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Activation of nanodroplets in 3D cell culture
For preliminary validation of the potential utility of dye-loaded PFC nanodroplets in biological studies, we generated 3D collagen matrix with embeded 4T1 mouse breast cancer cells expressing nuclear H2B-mCherry and Lifeact-GFP detecting actin filaments [55]. Culture media mixed with ND-Cy3 was incubated with the 3D cell culture before multiphoton imaging. Similar to our method of imaging phantom and suspension samples, 3D cell cultures were initially imaged using 1280 nm laser excitation (0-52 seconds) to confirm the absence of non-specific induction of THG ( Fig. 6(a)) followed by additional exposure to 1090 nm excitation for nanodroplet activation (during 52-100 seconds in the imaging sequence). We observed time dependent activation of the nanodroplets, with the emerging THG signals adjacent to the cells between 52 to 100 seconds of 1090 nm excitation ( Fig. 6(b) and Visualization 3). Most of the microbubbles appear to form extracellularly (see arrows in Figs. 6(b) and 6(c)), although, we cannot exclude the possibility of some nanodroplets been also endocytosed by cells. As in the case of PAA phantoms, the initially formed microbubbles underwent a significant size expanson over time after 100 seconds despite removal of the 1090 nm laser (Figs. 6(c)-6(f) and Visualization 3). There were apparent coalescence events contributing to growth of the microbubbles (Figs. 6(d)-6(f) and Visualization 3). Differences in the expansion of microbubbles are most likely asociated with local non-uniformity of nanodropet distribution in the cell culture as well as non-uniformities in activation of liquidgas phase transition of PFC nanodroplets due to differences in their dye loading and sizes. The microbubbles with bigger initial sizes can undergo faster growth due to the Oswald ripening effect and coalescence of bubbles and/or bubbles with droplets as described in Section 3.2. THG signals were confined to the activated area demonstrating controlled spatial activation ( Fig. 6(g)). Fate-tracing of activated nanodroplets indicates interactions with cell surfaces and potentially integration of the plasma membrane of the adjacent cells into coating of the growing microbubbles (Figs. 6(d)-6(f) and Visualization 3).
Notably, the mCherry signal inside the activated area was diminished after exposure to photoactivation, suggestive of photobleaching ( Fig. 6(g), dashed box). To test this assumption we carried out an experiment where mCherry expressing cells were labeled with calcein AM vital dye and were exposed to 1090 nm laser at 102 mW for 150 seconds to mimick conditions of nanodroplet activation used in our studies (data not shown). A continuous decay in the mCherry signal was observed with no decay in the calcein AM fluorescence indicating photobleaching of mCherry label whithout photo-cytotoxicity.   Fig. 7(e)). D oplet activation intact plasma ated with 1100 280 nm. Subs d 1280 nm, res f cells 1 and 3 the cell plasm Fig. 7(d) [12,13,65,66].
The effects of microbubble expansion on cell viability in direct vicinity may pose challenges in tissue imaging applications, but also open avenues for other applications. To control cell damage, we currently focus on minimizing the expansion of activated microbubbles starting with achieving a more uniform distribution of nanodroplets in cell cultures and optimizing nanodroplet concentration. For interventional purposes, we envision, for example, increasing permeation of blood vessels, e.g. in blood-brain barrier, via precise disruption of endothelium under THG monitoring. A similar concept was recently reported using microbubble-assisted focused ultrasound [67]. We thus foresee that the spatial precision of two-photon-mediated delivery as a tool in pre-clinical studies may offer particular opportunities for localized delivery. Likewise, locally triggered disruption of cancer cells may be exploited for release of cancer antigens for immunotherapy [68,69]. Finally, due to high level of interest in applications of PFC nanodroplets in photoacoustic and ultrasound imaging [38,[70][71][72], it is important to have tools that would allow a high resolution longitudinal imaging of PFC nanodroplet activation in biological samples similar to our study presented here.
Potential applications of the two-photon activatable phase-change nanodroplets could be greatly facilitated by the pioneering work of Professor Tuchin in the area of tissue optical clearing (TOC) [73][74][75][76][77]. This method is based on modifying refractive index of biological tissues that leads to reduction of tissue light scattering and making biological specimen more transparent [78]. The combination of multiphoton microscopy and TOC was shown to achieve depth of penetration in biological tissues exceeding 1000-2000 µm [79,80]. Therefore, application of TOC could significantly increase the depth in tissue where presented here activatable THG agents can be excited and imaged, thus extending the range of their potential applications.

Disclosures
The authors declare that there are no conflicts of interest related to this article.