The results obtained from the single particle experiments provide a coherent picture of bubble formation. We observed a fast transient decrease in the intensity of the transmitted HeNe laser after irradiating a single nanoparticle with a laser pulse of a radiant exposure exceeding 60 mJ/cm2
. The duration of this intensity decrease marks the lifetime of the laser induced vapor bubble. It shows a symmetric dependence on time, consistent with the temporal evolution of the transient vapor bubble size [18
]. A change in the index of refraction due to heating of the water, on the other hand, would have caused an asymmetric signal shape with a steep initial slope and an exponential relaxation due to the heat dissipation by diffusion [19
]. Additional second and third transient intensity drops that occasionally appeared at high radiant exposures with much smaller amplitude visualized the occurrence of bubble rebounds [20
By using the pressure transducer, we detected two distinct pressure transients upon irradiation, whose time delay perfectly coincided with the length of the HeNe laser transmission drop. Interestingly, the transients are of the same polarity and their shape does not change with their mutual delay, which means that they are not related to volume changes of the bubble. Instead, the pressure transients are associated with the initial and final stages of the expansion and collaps, which are known to create strong acoustic transients [13
]. We further noticed that the peak-to-peak amplitudes of both transients increased proportionally to the bubble lifetime (see ) while their ratio approaches unity with increasing bubble lifetime (not shown). The sequence of two pressure transients of similar amplitude, which represent a distinct signature of a transient vapor bubble, therefore allows us to identify the occurrence of single vapor bubbles in the cell experiments.
Flash photography of the bubbles further enabled us to relate the lifetime of a vapor bubble to its size. The bubble radius obtained from an image and the bubble radius calculated from the measured lifetime using Eq. (1)
nicely match (see ). The fact that the measured radius was about 20% smaller than expected based on the lifetime measurements and Rayleigh's equation is probably a consequence of the bubbles being in contact with the glass interface. Rayleigh's equation was derived for free spherical bubbles and may not be strictly applicable to our case. It has been observed before that the expansion and collapse of a bubble close to a surface are damped compared to one in free liquid [24
]. This can explain the lower measured radius compared to the calculation from the lifetime. Another reason for the slight discrepancy between the measured and the calculated bubble lifetime might be caused by the uncertainty to precisely determine the maximum bubble expansion from the flash photography images. Taking this into account our results from cw laser probing and flash photography of the bubbles show very good agreement.
These experiments demonstrate that we can detect vapor bubbles around single nanoparticles using two different methods and estimate their size from measured lifetimes. The HeNe laser probing provides a sensitive tool capable of measuring vapor bubble lifetimes as short as 10 ns which correspond to a vapor bubble diameter of around 110 nm calculated with Eq. (1)
. Whereas the optical method requires a probe laser in a transmission setup, the acoustic measurement is basically applicable from any direction. This made the pressure measurement more flexible and easier to implement in our microscopic setup for cell experiments.
Since a boundary layer close to a nanoparticle may influence vapor bubble nucleation [25
] or facilitate local melting of the particle [26
], experiments to measure the vapor bubble formation threshold and pressure transients were also performed with particle suspensions.
In the nanoparticle suspension vapor bubbles were detected at radiant exposures above 80 mJ/cm2 by brightfield flash photography. This method is less sensitive than the focused laser probing on the single particles and therefore yields just an upper limit for the threshold.
The pressure measurements showed transients consistent with bubble formation at 60 mJ/cm2. This was found by estimation of the pressure amplitude in the irradiated suspension based on results from the measurements with single particles:
The amplitude Â
of the transducer signal is proportional to the pressure transient which is the sum of the transients
from all N
irradiated particles in the cuvette.
can be deduced from the proportionality of pressure amplitude and lifetime known from and from a function
approximated to the data shown in which describes the dependency of the lifetime on the radiant exposure. This is summarized in Eq. (2)
denotes the radiant exposure at the site of the n-
th particle. The integration over particles can be replaced by an integration over the volume.
The radiant exposure
in Eq. (3)
is given by the pulse energy E
and the beam profile
, whereas the particle concentration dn/dV
can be regarded as constant, yielding
The beam profile
was measured with a CCD camera. The pulse energy E
was used as a parameter. Equation (4)
can be rewritten asEquation (5)
was then fitted to the experimental data by varying the constant. For the visualization and comparison with experimental data, the pulse energy is substituted by the peak radiant exposure
The result is shown in as a solid line. It shows good agreement with the measurement and thus indicates that our calculation is based on a sensible assumption for pressure transient generation, which means that the transients in fact stem from formation and collapse of vapor bubbles.
The same result is obtained when Â(H)
is calculated based on a theoretical approach reported by Egerev et al. [13
]. As a simplification of their theoretical deduction of the pressure amplitude caused by vapor bubble formation, they found a linear dependency of the pressure amplitude on the radiant exposure, H,
exceeding the bubble formation threshold Hth
. The dotted line in shows the result of this calculation in which
is used instead of
with a bubble formation threshold Hth
of 60 mJ/cm2
. It also matches the experimental results very well thus justifying the assumed radiant exposure threshold Hth
for bubble formation.
Hence we have shown that measurements on single particles and on particles in suspension yielded the same value of 60 ± 5 mJ/cm2 for the vapor bubble formation threshold. Therefore we conclude that the solid boundary in the single particle experiment, though it may have influenced the bubble lifetime, had no significant influence on the bubble formation threshold.
After investigating vapor bubble formation close to the surface plasmon resonance wavelength we extended our research to near infrared wavelengths that are more interesting for certain biological applications. Data on off-resonant excitation is hard to find in literature, probably since it is considered inefficient because of the low absorption cross-section. This expectation is based on the idea that vapor bubble formation is a thermal process, which means that the heat absorbed in the particle is transferred to the water raising its temperature up to the point of evaporation. If vapor bubble formation worked like that, using the same laser at different wavelengths the same energy deposition
should have the same effect.
Our data () indeed shows an increase of the thresholds upon departure from the resonance, but surprisingly, the threshold radiant exposure did not change by the same factor as the absorption cross-section when changing the irradiation wavelength. This indicates that vapor bubble formation is not only a thermal process. We suspect that also the local field strength affects bubble formation and are currently working on the theoretical explanation.
Our primary interest is the application of vapor bubble formation for medical therapy. Based on the presented results we assume that the threshold for bubble formation around a single nanoparticle is not heavily influenced by the properties of biological tissue and therefore is expected to be around 60 mJ/cm2 as well when irradiating at 532 nm. Considering the fact that vapor bubbles generated at a radiant exposure close to the formation threshold are probably far too small to damage the targeted cells, we expect even higher values for the damage threshold of cells.
Our experiments with macrophages that had been incubated with the same nanoparticles, though, yielded even lower thresholds for acute cell damage than the single particle vapor bubble formation thresholds. It seems reasonable to assume that this difference is caused by accumulation of particles in lysosomes in the cells. In TEM images of Bac-1 cells we found large lysosomes of up to micrometer size depending on the amount of nanoparticles available during incubation. It has been pointed out that aggregation of particles inside cells lowers the damage threshold [27
] which apparently happened here.
Further information on the damage process was gained from pressure measurements during irradiation. The recorded pressure signals showed two separate transients of identical shape and often of similar amplitude, especially for high radiant exposures. As seen in the single particle experiments such transients are signs of bubble formation and collapse. A detailed examination of the delay between the two transients revealed that cell damage always occurred for bubble diameters exceeding about half the diameter of the cell.
The lowest threshold for cell damage upon 532 nm irradiation we found was about 40 mJ/cm2
for cells incubated with the highest nanoparticle concentration. This value is smaller than results previously reported in the literature for similar experiments. Lapotko et al. [4
] reported cell damage above 1 J/cm2
and bubbles in tumor cells at radiant exposures of 100-300 mJ/cm2
using 30 nm spherical gold particles. Zharov et al. [14
] suggested induction of bubble formation around 30-40 nm gold spheres at high particle density and a radiant exposure of 500 mJ/cm2
to damage cells.
We guess that the strong accumulation of nanoparticles in the macrophages are the key factor that allows for the lower damage threshold. High particle numbers within a small volume can cause a large temperature rise even at relatively low radiant exposures. Evidence for this and a more detailed discussion of the accumulation effects will be provided in a forthcoming publication.
However, lowering the vapor bubble formation threshold might not be the only effect of the particle accumulation. Since it has been reported before that plasmon resonances of particle aggregates were shifted towards the near infrared compared to the single particles [28
], it could be advantageous to irradiate the Bac-1 cells at longer wavelengths. Therefore we also performed the cell irradiation experiments at 600 nm and 755 nm with cells incubated at the highest nanoparticle concentration of 4.5
particles/ml. At 600 nm the damage threshold (45 mJ/cm2
) was practically the same as for 532 nm. At 755 nm the threshold was found to be about 130 mJ/cm2
. Vapor bubbles at the damage thresholds were found to be of the same size as observed at 532 nm. The similarity of the thresholds measured for 532 nm and 600 nm may be a result of a shift in the absorption peak but cannot be regarded as significant considering that the threshold values for vapor bubble formation around single particles were also quite similar. However, the damage threshold in the NIR is surprisingly low. Whereas at 532 and 600 nm the damage threshold is 75% of the single particle bubble formation threshold it is 35% at 755 nm. That means that like observed when irradiating single particles vapor bubble formation is also more efficient in the near infrared when irradiating accumulated particles. Heat deposition or bubble nucleation or maybe both are apparently enhanced at longer wavelengths.
These effects should be taken into account when designing a medical application with any type of nanoparticles. However, more research is necessary to clarify these issues for other nanoparticle-laser systems.