2.1.

Nanoparticle Characterization

Gold nanospheres (20-, 40-, 60-, 80-, 100-nm diameter) with resonance peaks near 532 nm were purchased from Cytodiagnostics (Burlington, Ontario, Canada). Gold nanorods with the same resonance peak (850 nm) and different diameters (10, 25, 40 nm) were obtained from Nanopartz Inc. (Loveland, Colorado). The actual nanorod dimensions were as follows: 45-nm length, 10-nm width, 4.5 aspect ratio (AR); 93-nm length, 25-nm width, 3.7 AR; 160-nm length, 44-nm width, 3.6 AR. NanoHybrids Inc. (Austin, Texas) supplied polyethylene glycol (PEG)-coated and silica-coated gold nanorods with 850-nm resonance. Gold nanorods with citrate and PEG surfaces at 800-nm resonance, gold nanoshells with different resonances (660, 800, 980 nm) and surface coatings [polyvinylpyrrolidone (PVP), lipoic acid (LA), and PEG] were procured from nanoComposix (San Diego, California). The 660-nm resonant nanoshells are 158 nm in diameter, with a silica core of 88- and 35-nm-thick gold shell. The 800-nm resonant nanoshells have a diameter of 154 nm, with a 120-nm core and 17-nm shell. The 980-nm resonant nanoshells are 230 nm in diameter with a core of 198- and 16-nm shell. Transmission electron microscopy (TEM) micrographs were recorded on a JEM-1400 (JEOL, Inc.; Peabody, Massachusetts) operating at an accelerating voltage of 80 kV. Samples for TEM were prepared by drop-casting $7\mu \mathrm{L}$ of nanoparticle solution onto a Formvar-carbon-coated copper grid (FCF-400Cu, Electron Microscopy Sciences; Hatfield, Pennsylvania) and allowing it to dry. Optical extinction spectra were collected using a dual-beam UV/VIS/NIR spectrophotometer (Lambda 1050, PerkinElmer; Waltham, Massachusetts).

2.2.

Multimodal Microscopy Setup

Nanobubble generation and detection were performed on a multifunctional microscopy platform incorporating pump-probe imaging, integrated scattering response, and acoustic detection (Fig. 1). For nanospheres, bubbles were generated by a pump pulse from a frequency-doubled, Q-switched Nd:YAG laser (532-nm wavelength, 5-ns pulse duration, Surelite I-10 with SSP-2, Continuum; San Jose, California) directed into the backport of an inverted microscope (IX71, Olympus; Center Valley, Pennsylvania). To excite nanorods and nanoshells with NIR absorption peaks, an optical parametric oscillator pumped with a third harmonic Nd:YAG (355-nm pump, 5-ns pulse duration, Surelite OPO Plus with SL III-10, Continuum) was used to deliver pump pulses, tuned to the nominal resonance peak wavelength of each sample over a range of radiant exposures. Pump-probe images were acquired on a CMOS camera (DCC3240M, Thorlabs; Newton, New Jersey) using a delay generator (Stanford Research Systems DG535; Sunnyvale, California) to precisely adjust the timing of a 575-nm-wavelength probe pulse from a nitrogen dye laser (MNL 205, Rhodamine 6G laser dye, Lasertechnik Berlin; Berlin, Germany) that illuminated the sample from above at a 45-deg angle. The integral scattering response of the generated bubbles was monitored by measuring the transmitted intensity of a 632.8-nm wavelength He–Ne laser (Model 117A, Spectra-Physics; Santa Clara, California) through the sample with a photodiode (APD110A, Thorlabs) on a digital oscilloscope (TDS2024C, Tektronix; Beaverton, Oregon). Acoustic transients were detected with a miniature 10 MHz, 2-mm diameter planar immersion ultrasonic transducer (XMS-310, Olympus IMS; Waltham, Massachusetts) placed in the sample chamber, using a digital oscilloscope (TDS2014, Tektronix) after 40 dB preamplification (5678 preamplifier, Olympus IMS).

Fig. 1

Schematic representation of the microscope setup.

2.3.

Data Analysis and Simulations

Data analysis was performed using Python 2.7 with the Anaconda Python distribution. Absorption cross sections of nanospheres and nanoshells were calculated from Mie theory with MiePlot v4.6. Absorption cross sections of nanorods were computed using the discrete dipole approximation code ADDA 1.3b4.²⁶ Bubble generation thresholds were determined using all three modalities on the microscope setup. Fifty pulses were used to irradiate each sample over a range of radiant exposures and the bubble response was recorded. Any pump-probe bubble image was recorded as a positive bubble response. If no bubbles were seen in the image but a scattering and acoustic response were both observed, this was also considered as a positive response. These binary data were plotted against their corresponding radiant exposures, and a logistic regression was performed. Owing to the stochastic nature of the bubble generation process, bubble threshold was defined as the 50% probability of bubble formation determined from logistic regression.²⁷

A reference table for the per-pulse ANSI MPEs for skin exposure at wavelengths used in this study is shown in Table 1.¹⁵

Table 1

MPE values for skin at 10 Hz pulse repetition rate over various wavelengths used in this study.

3.1.

Nanosphere Bubble Thresholds

To study the bubble generation phenomena associated with pulsed laser–nanoparticle interaction, we built a multimodality microscopy platform for pump-probe imaging, integrated scattering response, and acoustic response detection (Fig. 1). Each of the three detection methods has its own advantages and disadvantages. Pump-probe imaging is the most sensitive technique, allowing observation of any bubbles generated within the focal plane of the objective, although bubbles outside of the depth of focus or smaller than the optical diffraction limit will not be detected. Since a specific pump-probe delay must be selected to capture an image, this method cannot provide information about the bubble lifetime. The integrated scattering response provides a method for estimating bubble lifetime, as bubble formation and collapse scatter the He–Ne probe beam and produce a decrease in sample transmittance. Measured transmittance is affected by any bubble generated within the probe beam path, not just those within the beam focus. The disadvantage of this technique is that it has low sensitivity and may measure the response due to multiple bubbles even though only a single bubble is imaged. Acoustic transient detection is advantageous for detection of bubbles in turbid media, where the pump-probe and scattering response would be unusable, but the shortest detectable bubble lifetime is restricted by the acoustic transducer impulse response, which has a duration inversely proportional to the transducer bandwidth, that may be longer than a bubble lifetime. Therefore, the duration of the acoustic transient measurement only provides an upper bound for the bubble lifetime. Another disadvantage of the acoustic response detection is that acoustic transients due to thermoelastic expansion and cavitation have different amplitudes but similarly shaped waveforms, making it difficult to determine whether bubbles are being generated based on the acoustic signal alone.

Examples of the data obtained with the different modalities are shown in Fig. 2. A submicron bubble appears as a bright dot in the pump-probe image [Fig. 2(a)]. The integral scattering response [Fig. 2(b)] has a duration of $\sim 150\mathrm{ns}$, which corresponds to the bubble lifetime. The acoustic response of the generated bubble is shown in Fig. 2(c), starting at $\sim 350\mathrm{ns}$. The secondary waveform observed around 800 ns is due to the reflection off the wall of the sample chamber. Note that the time scales in Figs. 2(b) and 2(c) are not absolute as the oscilloscopes were triggered independently.

Fig. 2

(a) Representative pump-probe image, (b) integral scattering response, and (c) acoustic response of a nanobubble.

TEM micrographs of different diameter nanospheres used in this study are shown in Fig. 3(a). Figure 3(b) shows an example of the probability curves that were used to determine bubble thresholds. As seen in Fig. 3(c) and Table 2, there is a large decrease in bubble threshold as nanosphere diameter increases from 20 to 60 nm, and a much slower change from 60 to 100 nm. The experimentally determined bubble thresholds scale inversely with their absorption cross sections ($C_{\mathrm{abs}}$). The shape of this curve is similar to what has been reported in the literature, although there is still debate as to whether there should be a local minimum at 60 nm (above this size the absorption cross section is no longer proportional to the particle volume).^{11,12,14,19,28} These results also follow the same trend with particle size that we have observed for nanosphere damage thresholds.²⁹

Fig. 3

(a) Representative TEM micrographs of the nanosphere samples. (b) Example of the bubble generation probability curves that are used to determine the bubble thresholds. (c) Bubble generation thresholds as a function of particle diameter (circles, left axis) and their corresponding inverse absorption cross sections (triangles, right axis).

Table 2

Tabulated nanosphere bubble thresholds.

3.2.

Nanoshell Bubble Thresholds

3.2.1.

Effect of resonance peak

TEM micrographs of the nanoshells are shown in Fig. 4(a). All three samples were found to have similar bubble generation thresholds [Fig. 4(b) and Table 3] when excited at their plasmon peak. A previous report by Ogunyankin et al.²² using hollow gold nanoshells with picosecond laser pulses noted similar behavior. The fact that the bubble thresholds do not scale with the inverse of their $C_{\mathrm{abs}}$ suggests that other factors, such as shell thickness or overall particle size, influence the bubble generation process. While all three bubble generation thresholds were similar in magnitude, the radiant exposures required for 660- and 800-nm resonant shells ($36\pm 4$ and $40\pm 7\mathrm{mJ}/{\mathrm{cm}}^2$; Table 3) were higher than the ANSI MPE for skin at those wavelengths (20 and $31.7\mathrm{mJ}/{\mathrm{cm}}^2$; Table 1). The 980-nm resonant shells were found to have a threshold value ($35\pm 7\mathrm{mJ}/{\mathrm{cm}}^2$; Table 3) of about half the MPE ($72.6\mathrm{mJ}/{\mathrm{cm}}^2$; Table 1). It is important to note that in the case of the 660- and 800-nm resonant shells, scattering in tissue may produce fluences that reach the determined threshold levels even when the incident radiant exposure is at or below the MPE.³⁰ This indicates that there is a significant potential for bubble generation when using gold nanoshells combined with diagnostic-level pulsed laser irradiation in vivo.

Fig. 4

(a) TEM micrographs of the nanoshells with different resonance peaks. (b) Bubble generation thresholds for the three different nanoshells (bars, left axis) and the inverse of their calculated absorption cross sections (triangles, right axis). Error bars denote standard deviations. (c) Normalized absorbance spectra of the nanoshell samples.

Table 3

Bubble thresholds for nanoshells with different resonance peaks.

Nanoshell resonance (nm)	660	800	980
Bubble threshold (mJ/cm2)	$36\pm 4$	$40\pm 7$	$35\pm 7$

3.2.2.

Effect of surface modification

A variety of surface modifications can be used to impart nanoparticles with different physical and chemical properties. There is currently limited information on whether these modifications have any impact on plasmonic nanobubble generation. In the case of thiolated surface modifications, such as lipoic acid and PEG, they are known to detach from the particle surface under laser exposure and are not expected to impact the bubble generation process.³¹ We have compared bubble thresholds for 800-nm resonant nanoshells modified with a physisorbed polymer (PVP), and monodentate (PEG), and bidentate (LA) thiols [Fig 5 and Table 4]. As shown, similar thresholds were found, regardless of the surface modification, which is again in agreement with Ogunyankin et al.,²² who found similar thresholds for both citrate and PEG-modified hollow nanoshells.

Fig. 5

(a) TEM micrographs of the nanoshells with different surface modifications. (b) Bubble generation thresholds for the three different nanoshells. Error bars denote standard deviation. (c) Normalized absorbance spectra of the nanoshell samples.

Table 4

Bubble thresholds for nanoshells with different surface modifications.

Nanoshell surface	PVP	LA	PEG
Bubble threshold (mJ/cm2)	$40\pm 7$	$34\pm 6$	$39\pm 7$

3.3.

Nanorod Bubble Thresholds

3.3.1.

Effect of nanorod size

Gold nanorods are commonly used with a number of different optical techniques due to the wide tuning range of their plasmon resonance. One interesting feature of nanorods is that different-sized particles can have the same resonance peak by tuning the AR.³² To study the effect of nanorod size on bubble thresholds, we used nanorods with widths of 10, 25, and 40 nm, all with ARs resulting in plasmon peaks near 850 nm. The effective radii of these nanorods (radius of a volume equivalent sphere) are almost equivalent to their denoted widths. Bubble generation threshold decreased with increasing particle size, and roughly scaled with the inverse of $C_{\mathrm{abs}}$ [Fig. 6, Table 5]. Nanorod damage thresholds have been shown to display the same trend, which is attributed to less-efficient heat transfer to the medium as the surface-to-volume ratio decreases.³³ For the smallest nanorod size (10-nm width), the bubble generation threshold ($350\mathrm{mJ}/{\mathrm{cm}}^2$; Table 5) was about an order of magnitude higher than those obtained for nanoshells (34 to $40\mathrm{mJ}/{\mathrm{cm}}^2$; Table 3), whereas the 40-nm width nanorods had a comparable threshold ($43\mathrm{mJ}/{\mathrm{cm}}^2$; Table 5). This indicates that smaller nanorods are less efficient for bubble generation due to faster heat dissipation in the surrounding medium.

Fig. 6

(a) TEM micrographs of the nanorods with different surface modifications. (b) Bubble generation thresholds for the three different nanorods (bars, left axis) and the inverse of their calculated absorption cross sections (triangles, right axis). Error bars denote standard deviation. (c) Absorbance spectra of the nanorod samples.

Table 5

Bubble thresholds for nanorods with different widths at the same resonance peak.

Nanorod width (nm)	10	25	40
Bubble threshold (mJ/cm2)	$350\pm 80$	$120\pm 30$	$43\pm 9$

3.3.2.

Effect of surface modifications and coatings

The effect of surface modification on nanorod bubble thresholds was studied using citrate and PEG functionalized nanorods with an 800-nm resonance peak (Fig. 7). As seen in Fig. 7(b), there was no significant difference in the determined bubble thresholds with citrate ($630\pm 90\mathrm{mJ}/{\mathrm{cm}}^2$) or PEG ($750\pm 110\mathrm{mJ}/{\mathrm{cm}}^2$) surfaces. This was expected due to the detachment of surface ligands from the nanorod surface under laser irradiation.

Fig. 7

(a) TEM micrographs of the nanorods with different surface modifications. (b) Bubble generation thresholds for the two nanorods. Error bars denote standard deviation. (c) Absorbance spectra of the nanorod samples.

Silica (${\mathrm{SiO}}_2$) coating has been used to improve nanorod stability for photoacoustic imaging and has also resulted in a threefold increase in photoacoustic intensity.³⁴ The proposed mechanism for this enhancement is improved heat transfer to the surrounding medium—where the pressure wave originates—due to decreased thermal interface resistance. Since more rapid heating of water should impact the onset of vaporization, we evaluated the effect of silica coating on nanorod bubble formation thresholds. A comparison of results obtained during irradiation of gold nanorods with PEG and silica coatings is shown in Fig. 8. The silica-coated particles were found to have a significantly lower bubble threshold ($190\pm 40\mathrm{mJ}/{\mathrm{cm}}^2$) than the PEGylated ones ($750\pm 160\mathrm{mJ}/{\mathrm{cm}}^2$). Although the silica coating produced a notable reduction in the bubble generation threshold, the radiant exposure required still exceeds the MPE by a factor of 5.

Fig. 8

(a) TEM micrographs of the nanorods with and without silica coating. (b) Bubble generation thresholds for the two nanorods. Error bars denote standard deviation. (c) Absorbance spectra of the nanorod samples.

3.4.

Theoretical Analysis of Nanoparticle Heating

While gold nanospheres showed good agreement between their $C_{\mathrm{abs}}$ and bubble thresholds, the nanoshells and nanorods exhibited notable discrepancies. The nanorods with widths of 25 and 40 nm have similar $C_{\mathrm{abs}}$ but $\sim 3\times$ difference in bubble thresholds. This may be due to differences in the thermal relaxation times between the sizes of nanorods. Using the spherical volume-equivalent effective radii of these two nanorods, their thermal relaxation times were calculated according to Pustovalov et al.

In the case of the nanoshells, the thermal relaxation times were calculated as

As shown in Fig. 9, the inverse of the normalized $C_{\mathrm{abs}}$ now only varies by $\sim 15\%$ between the three nanoshells, similar to the difference in bubble thresholds observed experimentally. Although the trend in the scaled $C_{\mathrm{abs}}$ and bubble thresholds are not the same, the relative magnitude of these variations falls within the range of experimental uncertainty.

Fig. 9

Nanoshell bubble thresholds (bars, left axis) compared with the inverse of their $C_{\mathrm{abs}}$ after normalization to the thermal relaxation time and resistivity (triangles, right axis).

3.5.

Summary of Findings

Gold nanosphere bubble thresholds were found to be in the same range of radiant exposures as other experimental reports, validating our experimental approach.^11,28,37 The change in bubble threshold with nanosphere size is inversely correlated with the absorption cross section of the particles and suggests a thermo-mediated mechanism of bubble generation. All nanosphere bubble thresholds exceeded the MPE; therefore, the use of nanospheres with nanosecond lasers at current exposure limits is not expected to produce bubble generation. However, clustering of nanoparticles when applied in vitro or in vivo, which is known to reduce the bubble generation threshold, may allow for the production of bubbles below the MPE.¹¹ This effect is currently under investigation.

Gold nanoshells had the lowest bubble generation thresholds of the three particle shapes that were studied. In the case of 980-nm resonant shells, this threshold was about half the MPE for skin, indicating that bubble generation is a real possibility at “tissue-safe” exposures as would be used in diagnostic applications. Different surface coatings did not affect the bubble thresholds, which is to be expected due to detachment of surface ligands caused by pulsed laser exposure.³⁸ No correlation between the nanoshell absorption cross sections and bubble thresholds was observed, suggesting that other physical properties, such as the overall particle size or thickness or the gold shell, have a significant impact on the bubble formation process. The threshold radiant exposure levels for nanoshell bubble generation may also fall below particle damage thresholds. Nanoshells can be specifically designed for bubble generation below the damage threshold when exposed to off-resonant femtosecond pulses, but it remains to be seen whether this holds true in the nanosecond pulse regime.^19,20

Gold nanorods are known to be unstable under laser irradiation and may undergo melting and reshaping when exposed to nanosecond pulsed irradiation. This effect coupled with the high bubble generation thresholds observed in this study suggests that nanorods may not be an ideal choice of particle for in vivo bubble generation.³³ Increasing nanorod size showed a reduction in bubble threshold that roughly scaled inversely with the absorption cross section. The largest nanorod studied, with a length of 160 nm and width of 44 nm, had a bubble generation threshold similar to that of the nanoshells, but still slightly above the MPE. Silica coating was observed to significantly reduce the bubble threshold and can also improve photostability due to steric confinement of the gold, even if melting occurs.

In summary, gold nanoshells appear to be the preferable particle shape for bubble generation due to their thresholds being near or below the MPE. Previous work demonstrating the robustness of nanoshells under off-resonance femtosecond pulsed irradiation suggests that it may be possible to generate bubbles without damaging the nanoshells, alleviating concerns about harmful bioeffects due to nanoparticle melting and evaporation. Preliminary results from our group indicate that the damage threshold for the 660- and 980-nm resonant shells is higher than their bubble generation threshold, while the 800-nm resonant shells undergo damage prior to reaching the bubble threshold. Further studies are necessary to elucidate the effects of nanoparticle agglomeration in vitro and in vivo on bubble generation thresholds.