Biological applications where nanoparticles are used in a cell environment with laser irradiation are rapidly emerging. Investigation of the localized heating effect due to the laser irradiation on the particle is required to preclude unintended thermal effects. While bulk temperature rise can be determined using macroscale measurement methods, observation of the actual temperature within the nanoscale domain around the particle is difficult and here we propose a method to measure the local temperature around a single gold nanoparticle in liquid, using white light scattering spectroscopy. Using 40-nm-diameter gold nanoparticles coated with thermo-responsive polymer, we monitored the localized heating effect through the plasmon peak shift. The shift occurs due to the temperature-dependent refractive index change in surrounding polymer medium. The results indicate that the particle experiences a temperature rise of around 10 degrees Celsius when irradiated with tightly focused irradiation of ~1 mW at 532 nm.
©2011 Optical Society of America
Metal nanoparticles exposed to incident laser irradiation at wavelengths close to the surface plasmon resonance efficiently couple the optical energy and generate heat. The heat induced by the laser-irradiated metal nanoparticles is considered to be localized near the nanoparticles, and due to their small scale, becomes difficult to measure. The heating of nanoparticles in liquid environments where the nanoparticle temperature may rise significantly and then pass on the heat to molecules in the immediate vicinity is of particular interest since gold nanoparticles are entering wide use in biomedical fields. Emerging applications build on the biocompatible, or relatively nontoxic, nature of the particle, combined with some unique optical physics which allows plasmonic photothermal therapy, photoacoustic tomography, photothermal imaging, and surface enhanced Raman spectroscopy [1–7]. For the successful application of gold nanoparticles, the investigation of the localized heating effects must be carried out in order to firstly understand the influence on the biological samples, and subsequently preclude unwanted thermal effects. The difficulty of such local temperature measurement lies in the fact that a thermal contrast mechanism as well as a spatial resolution of several nanometers is necessary to selectively observe the near-field heating of the nanoparticle. Several techniques for nanoscale thermometry have been reported, such as nanolithographic, nanomaterial-based, fluorescent materials, and nanoscale superstructure thermometry method [8–12]. In these methods, temperature probes such as a silicon tip, carbon nanotubes, terbium doped silica glass and CdTe nanoparticles were used. The thermal effect of the probe itself, either as a light absorber, or heat sink/source may be comparable to that of single nanoparticles. As a result, those techniques need careful evaluation before being applied to the measurement of single gold nanoparticles. Nevertheless, several works have been reported on nanoscale thermometry for gold nanoparticles. In 2006, gold nanoparticles embedded in an ice matrix were observed by Raman spectroscopy . The sample was irradiated with laser and heat generation was inferred from the melting of the ice matrix. In 2008, the temperature elevation of gold nanoparticles was monitored using photoacoustic signals during photothermal therapy . Both measurements were successful with relatively low spatial resolution. We aim to improve the measurement spatial resolution to the surface temperature of gold nanoparticles and measure in a range approximately ~40 degrees, which is critical to many biomaterials such as proteins .
We have investigated the local temperature of a laser irradiated single nanoparticle by white light scattering spectroscopy. White light scattering spectroscopy is a powerful method to measure the dielectric constant of the nanomaterial . In our method, the temperature can then be estimated through the surface plasmon peak shift due to the refractive index change of the surrounding medium. Although it is possible to observe a temperature-dependent spectral peak shift in uncoated nanoparticles at temperatures of 100 degrees or higher, the range of temperature measurement of interest in our experiment (approximately room temperature) does not allow the observation of a peak shift with uncoated particles. Gold nanoparticles were therefore coated by thermo-responsive polymer, poly(N-isopropylacrylamide) (pNIPAM) so that the surrounding medium was sensitive to nanoparticle temperature. The heating effect was monitored by measuring the peak scattering wavelength derived from the surface plasmon of gold nanoparticles, which varied with the refractive index of the surrounding polymer. The scattering occurs predominantly from the gold-polymer interface, allowing a measurement of the gold nanoparticle temperature, and the gold nanoparticle temperature is assumed to be homogenous in the nanoparticle due to thermal conductivity. For nanoscale temperature measurement, pNIPAM-coated gold nanoparticles were fixed on a glass cover slip, immersed in water, and illuminated by continuous laser. The sensitivity of the measurement results from the scattering spectra sensitivity to the refractive index change at the boundary of the metal surface and coated polymer. The measurement spatial resolution is then predominantly limited by the size of the nanoparticle itself, which in these experiments was 40 nm. Temperatures were calibrated by then using a temperature controlled cell to heat a bulk sample of nanoparticles and measuring the temperature dependent peak shift by UV-VIS absorption spectroscopy. The heating effect of single gold nanoparticles under different laser powers and wavelengths are measured and discussed.
2. Calculated scattering property and heat generation of a gold nanoparticle
White light scattering spectroscopy for local temperature measurement is based on the plasmonic property of metal nanoparticles, and the scattering efficiency of gold nanoparticles was calculated on the basis of Mie theory . Scattering, extinction, and absorption efficiency, Qsca, Qext, Qabs, are described as by the following series:18].
Calculations were performed for different nanoparticle sizes including diameters of 40, 50, 60, and 70 nm. To first evaluate the nanoparticle size dependent scattering spectra, the surrounding media was set to water, with a refractive index of 1.33 + 0i. Figure 1(a) shows calculated absorption spectra for a gold nanoparticle with the diameter of 40 nm and with the surrounding refractive index of 1.33. In Fig. 1(a), blue dotted lines indicate illumination wavelengths, 488 and 532 nm. Figure 1(b) shows the calculated scattering spectra of different sized gold nanoparticles. The maximum value is then taken to be the scattering peak wavelength. As shown in Fig. 1(b), the peak wavelength changes from 533 nm to 546 nm as the particle size increases.
In the case of a pNIPAM-coated gold nanoparticle, the real part of the surrounding refractive index varies from 1.33 to 1.42 , whereas the imaginary part is always 0. The scattering spectra calculated for a gold nanoparticle surrounded with different refractive indices are shown in Fig. 1(c). A shift of around 8 nm was observed in Fig. 1(c). It has been reported that the refractive index of pNIPAM depends on the temperature, therefore, we expected that by monitoring the scattering peak shift, we can measure the local temperature of the particle .
3. Coating gold nanoparticles with poly(N-isopropylacrylamide)
pNIPAM-coated gold nanoparticles were synthesized by the procedure shown in Ref. 20. pNIPAM-coated gold nanoparticles were fixed on an aminosilane-modified glass cover slip by drop coating. Figure 2(a) and 2(b) show scanning electron microscope (SEM) images of a pNIPAM-coated gold nanoparticle and an uncoated gold nanoparticle on a silicon substrate. The pNIPAM coating appeared with lower contrast around the solid gold core. Judging from the images, the typical size for the gold core was ~40 nm and the pNIPAM layer thickness was approximately 20 nm.
4. Optical setup for white light scattering spectroscopy
The optical setup for measuring nanoparticle scattering spectra and irradiating gold nanoparticles by laser is shown in Fig. 3 . The white light source for scattering spectra was a xenon lamp and was spatially filtered through a 50 μm pinhole. The light was collimated and introduced to an objective lens (100x, oil immersion) of 1.35 NA. Dark field illumination was realized by inserting a mask and an iris in the incident path (to block NA<1) and detection path (to block NA>1). The scattering signal was collected by the same objective lens, introduced to a spectrometer and detected by a Peltier cooled CCD camera. According to the Rayleigh scattering image on the entrance slit of the spectrometer, we selected the area of signal to extract only the scattering from a single pNIPAM-coated gold nanoparticle.
The heating laser was provided by CW diode lasers of 488 nm and 532 nm wavelength. The incident power was adjusted by an electrically controlled variable ND filter. The incident laser was expanded by 5 times and overlapped with the white light source. The Rayleigh scattering of laser irradiation was cut by an edge filter before entering the spectrometer. The white light scattering measurement of a laser-irradiated gold nanoparticles was performed with 60 sec acquisition times under continuous laser irradiation. Laser power was measured at the sample focus.
5. Local temperature measurement of a laser-irradiated gold nanoparticle
For precise calibration of scattering peak shift to temperature increase, it is optimal to measure the calibration curve for each nanoparticle by observing how the scattering spectral peak shifts with temperature by measuring a single nanoparticle spectra while heating the entire sample holder on the microscope, however, it was not possible due to thermal drift. Instead, the bulk temperature of a water-based solution of pNIPAM-coated gold nanoparticles was measured by UV-VIS absorption spectrometer in a temperature-controlled cell to evaluate relationship between the temperature and the surface plasmon peak wavelength. The measurement was performed when the temperature-controlled cell had reached thermal equilibrium conditions.
Figure 4 shows the scattering peak shift response of pNIPAM-coated gold nanoparticles to the bulk temperature. Peak wavelengths were obtained by Gaussian curve fitting of raw data. pNIPAM has a phase transition at ~35 degrees c and therefore the refractive index change is most sensitive to changes near this temperature . Furthermore, it has been reported that the surface plasmon resonant peak wavelength is expected to undergo red shift with increasing temperature . As Fig. 4 shows, the extinction peak predominantly shifts from 538 nm to 552 nm as the temperature rises from 10 to 40 degrees c. This result is consistent with previous works and showing that pNIPAM-coated gold nanoparticles which we prepared can be used for monitoring the localized heating effect, with particular emphasis on the temperature range which is relevant to biological cells. Cell lipid bilayer stability is known to change significantly in this temperature range with a rise of only several degrees .
Following calibration, the local heating effect could then be measured for single gold nanoparticles under laser irradiation. pNIPAM-coated gold nanoparticles were fixed on a glass cover slip and immersed in water. A single pNIPAM-coated gold nanoparticle was irradiated with different laser powers, and scattering spectra were measured under each irradiating power. Figure 5(a) shows the spectra of a single pNIPAM-coated gold nanoparticle under 532 nm laser excitation, and Fig. 5(b) shows the relationship between the scattering peak wavelength and the laser power. In Fig. 5(a), the lowest spectrum shows an approximately Gaussian scattering curve. Since the peaks are close to the 532 nm excitation laser wavelength, spectra were generally taken with an edge filter which cut out a portion of the scattering spectra but left the peak intact, as shown in Fig. 5(a).
When the peak shifts are plotted against laser power in Fig. 5(b), the peak wavelength is observed to shift to progressively longer wavelengths as the laser power increased. Prior to laser irradiation, the scattering spectrum had a peak wavelength at 542 nm. This peak wavelength was longer than that in the calculated spectrum of 40 nm gold particles. Due to the measurement of single nanoparticles, a range of different initial scattering peak wavelengths were observed. Some differences are likely to be due to minor variations in coating thickness and the glass substrate on which the particle rests . When the laser irradiation started, the peak wavelength is seen shift to longer wavelengths, and by 1.2 mW, the peak reached 549 nm. Consequently, approximately 7 nm in total peak shift was observed. According to Fig. 4, a 7 nm peak shifts corresponds to a temperature increase of 10 degrees c. In this case, a single gold nanoparticle at room temperature (22 degrees c) was heated to 33 degrees c by a laser irradiation power of 1.2 mW. Such a small temperature increase at ~40 degrees c in the nanoscale region around the particle is very difficult to detect by conventional methods. We also note that the peak shift was caused by laser power rather than exposure time, since the nanoparticle can reach steady-state heating in a time scale of only nanoseconds . The measurement time (60 seconds) was much longer than the thermal equilibrium time due to the requirement of collecting enough scattering signal to measure a single nanoparticle spectra.
We observed several types of heating effect with 532 nm irradiation. In Fig. 6(a) , 5 curves from different gold nanoparticles were shown. Each curve had different starting wavelengths since there are different distances between the gold nanoparticles and the glass substrate, differences in coated layer thickness, or in particle size. The data shown in Fig. 6(a) reveal that the heating effects appear to be different for each single particle. This makes it difficult to accurately predict the thermal heating of nanoparticles by laser, though most of the spectra show a shift to longer wavelengths with increasing laser power. It seems likely that the local heat generated and dissipated by a single nanoparticle is sensitive to its size, shape and local environment. This kind of variability is hidden by a collective measurement of bulk temperature in nanoparticle samples, and shows the limitations on the ability to measure a particular temperature rise for a particular nanoparticle in, for example, a live cell under laser irradiation.
Even with the expected variability between particles which is highlighted by this measurement method, we still can predict tendencies of the laser heating effect. Figure 6(b) shows the scattering peak wavelength shift with increasing laser power under 488 nm laser irradiation. Compared to the 532 nm laser case, the 488 nm laser appears much less effective to heat a single gold nanoparticle. Most of the data exhibit almost no increase, though small fluctuations within ~2 nm are observable on close inspection. This suggests that ~3 mW was not strong enough to heat a single gold nanoparticle by 488 nm laser and plasmon absorption-based heating strongly depends on laser wavelength, as expected from Fig. 1(a). In the case of 532 nm irradiation, 4 out of 5 particles show a 10 degree C increment from the original temperature by a ~1 mW illumination intensity..
We discuss the effect of the surrounding environment and application of our data. Absorption of pNIPAM is much lower than that of gold nanoparticles in the visible range . As a result, absorption of light by gold nanoparticles is dominant in our experiment. When absorption of surrounding material is very low compared to gold in other experiments, our results can be similarly applied. On the other hand, in experiments where the surrounding materials absorb light, especially near 540 nm, the relative absorption by gold nanoparticles will be reduced and the assumption of nanoparticle-dominated absorption will break down so that it becomes difficult to apply our results to these experiments. Furthermore, when the thermal conductivity and refractive index of the surrounding materials are similar to that of pNIPAM for example, proteins and other biological molecules, our results can directly be applied. If, however, the conductivity and refractive index are significantly different from pNIPAM, further calibration will be required before estimating the temperature effects.
Absorption of pNIPAM and water is small compared to plasmonic absorption of gold nanoparticles, hence, the temperature rise is mainly related to the absorption of gold. pNIPAM-coated gold nanoparticles are on a glass substrate and the distance between individual particles and the substrate is different due to variations in the polymer thickness. Heat diffusion from the gold nanoparticles therefore varies between individual single gold nanoparticles. As a result, the equilibrium temperature is expected to vary and introduce some error in the measurements.
6. Implications for laser-irradiation of gold nanoparticles in cellular environments
It is worth summarizing what these experiments predict for the use of nanoparticles as tags or contrast agents in live cells with laser irradiation. There are a wide range of nanoparticle types, sizes, and laser excitation conditions in current use. The laser wavelengths used with nanoparticles are often longer than 532 nm, and this method will allow experimental determination of upper power limits involving laser irradiation and nanoparticles. While we have used 532 nm for the experimental measurements in this paper, 532 nm is near the peak absorption of 50 nm nanoparticles, which is the most common size for surface-enhanced Raman measurements. For this reason, the 532 nm results are of value to experiments using other laser wavelengths in that they should indicate an upper limit on the thermal effect. Other wavelengths in the visible or near infrared range should result in lower heating of the particle. The measurement method used here is also most sensitive to temperature changes around 35 degrees Celsius. While this is limiting in terms of wide temperature range measurements, it is ideal for experiments using cultured cells with laser heating of nanoparticles since the cell temperature is usually maintained at 37 degrees.
As evident in Fig. 5(b) and Fig. 6, the application of even low power ~0.01 mW resulted in a change in the scattering peak wavelength. By using the bulk temperature calibration data, we can estimate that the temperature can rise by several degrees around the nanoparticle. It is important to note that this measurement method results in very localized measurement of the temperature, on a scale of several tens of nanometers. Cells can survive brief exposure to localized plasma formation or complete ablation of sub-micron sized areas in the cytosol , and the nanoscale localized temperature increase is unlikely to manifest as an observable effect in the irradiated cell. Nevertheless it is necessary to understand if significant heating is occurring near the nanoparticle since this may affect protein folding, molecular binding, pH, or a number of other factors which may be of interest in the measurement.
In this work, we have successfully observed the behavior of nanoscale localized heating of a laser irradiated single gold nanoparticle by white light scattering spectroscopy. The temperature increment of approximately 10 degrees c was observed by irradiating a gold nanoparticle by 532 nm laser at ~1 mW. On the other hand, single gold nanopaticles irradiated by 488 nm did not display a measurable temperature rise, which is consistent with the notion that the heating is dominated by absorption of the incoming light at the surface plasmon resonance wavelength. Even laser irradiation of 0.01 mW or less appeared to produce a temperature dependent shift in the scattering spectra peak wavelength. This has implications for the use of nanoparticles in cellular environments and shows that the heating effect of laser light on nanoparticles may not be negligible, and should be further studied, particularly at wavelengths longer than 532 nm. It was also found that the local heating effects for each nanoparticle were not uniform and fluctuations were observed in both on-resonant and off-resonant cases. This indicates that the localized heating itself may be unstable and susceptible to the immediate environment. The effects of nanoscale heat conduction and convection flows over the particle may be responsible for some of the observed instability. Such high sensitivity temperature measurement for a nanoparticle at room temperature range together with nanometer spatial resolution has not previously been achieved. The method will be useful to estimate the local temperature effects of laser irradiated nanoparticles in a variety of materials including particles in an inorganic matrix and endocytosed or injected nanoparticles in living cells.
The authors would like to express gratitude to Dr. N. Takeyasu for providing useful advice. This work was supported by the Japan Funding Program for World- Leading Innovative R&D on Science and Technology (FIRST Program).
References and links
1. X. Huang, P. K. Jain, I. H. El-Sayed, and M. A. El-Sayed, “Plasmonic photothermal therapy (PPTT) using gold nanoparticles,” Lasers Med. Sci. 23(3), 217–228 (2008). [CrossRef]
2. A. Barhoumi, R. Huschka, R. Bardhan, M. W. Knight, and N. J. Halas, “Light-induced release of DNA from Plasmon-resonant nanoparticles: Towards light-controlled gene therapy,” Chem. Phys. Lett. 482(4-6), 171–179 (2009). [CrossRef]
4. X. D. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol. 21(7), 803–806 (2003). [CrossRef] [PubMed]
6. K. Fujita, S. Ishitobi, K. Hamada, N. I. Smith, A. Taguchi, Y. Inouye, and S. Kawata, “Time-resolved observation of surface-enhanced Raman scattering from gold nanoparticles during transport through a living cell,” J. Biomed. Opt. 14(2), 024038 (2009). [CrossRef] [PubMed]
9. C. M. Tan, J. Jia, and W. Yu, “Temperature dependence of the field emission of multiwalled carbon nanotubes,” Appl. Phys. Lett. 86(26), 263104 (2005). [CrossRef]
10. H. Aizawa, T. Katsumata, S. Komuro, T. Morikawa, H. Ishizawa, and E. Toba, “Fluorescence thermometer based on the photoluminescence intensity ratio in Tb doped phosphor materials,” Sens. Actuators A Phys. 101(1), 78–82 (2002).
11. J. Lee, A. O. Govorov, and N. A. Kotov, “Nanoparticle assemblies with molecular springs: a nanoscale thermometer,” Angew. Chem. 117(45), 7605–7608 (2005). [CrossRef]
12. C. Gota, K. Okabe, T. Funatsu, Y. Harada, and S. Uchiyama, “Hydrophilic fluorescent nanogel thermometer for intracellular thermometry,” J. Am. Chem. Soc. 131(8), 2766–2767 (2009). [CrossRef] [PubMed]
13. H. H. Richardson, Z. N. Hickman, A. O. Govorov, A. C. Thomas, W. Zhang, and M. E. Kordesch, “Thermooptical properties of gold nanoparticles embedded in ice: characterization of heat generation and melting,” Nano Lett. 6(4), 783–788 (2006). [CrossRef] [PubMed]
14. J. Shah, S. Park, S. Aglyamov, T. Larson, L. Ma, K. Sokolov, K. Johnston, T. Milner, and S. Y. Emelianov, “Photoacoustic imaging and temperature measurement for photothermal cancer therapy,” J. Biomed. Opt. 13(3), 034024 (2008). [CrossRef] [PubMed]
15. X. He, W. F. Wolkers, J. H. Crowe, D. J. Swanlund, and J. C. Bischof, “In situ thermal denaturation of proteins in dunning AT-1 prostate cancer cells: implication for hyperthermic cell injury,” Ann. Biomed. Eng. 32(10), 1384–1398 (2004). [CrossRef] [PubMed]
16. M. Y. Sfeir, F. Wang, L. Huang, C. C. Chuang, J. Hone, S. P. O’Brien, T. F. Heinz, and L. E. Brus, “Probing electronic transitions in individual carbon nanotubes by Rayleigh scattering,” Science 306(5701), 1540–1543 (2004). [CrossRef] [PubMed]
17. S. Kawata, Near-Field Optics and Surface Plasmon Polaritons (Springer, 2001).
18. P. B. Johnson and R. W. Christy, “Optical constants of the noble metals,” Phys. Rev. B 6(12), 4370–4379 (1972). [CrossRef]
19. B. W. Garner, T. Cai, S. Ghosh, Z. Hu, and A. Neogi, “Refractive Index change due to volume-phase transition in polyacrylamide gel nanospheres for optoelectronics and bio-photonics,” Appl. Phys. Express 2, 057001 (2009). [CrossRef]
20. R. Contreras-Cáceres, J. Pacifico, I. Pastoriza-Santos, J. Perez-Juste, A. Fernandez-Barbero, and L. M. Liz-Marzan, “Au@pNIPAM thermosensitive nanostructures: control over shell cross-linking, overall dimensions, and core growth,” Adv. Funct. Mater. 19(19), 3070–3076 (2009). [CrossRef]
21. R. M. J. Cotterill, Biophysics (Wiley, 2002). [PubMed]
22. T. Okamoto and I. Yamaguchi, “Field enhancement by a metallic sphere on dielectric substrates,” Opt. Rev. 6(3), 211–214 (1999). [CrossRef]
23. P. Keblinski, D. G. Cahill, A. Bodapati, C. R. Sullivan, and T. A. Taton, “Limits of localized heating by electromagnetically excited nanoparticles,” J. Appl. Phys. 100(5), 054305 (2006). [CrossRef]
24. J. Hofkens, J. Hotta, K. Sasaki, H. Masuhara, and K. Iwai, “Molecular assembling by the radiation pressure of a focused laser beam: poly(N-isopropylacrylamide) in aqueous solution,” Langmuir 13(3), 414–419 (1997). [CrossRef]