“Fluorescence vector probes and polarization-sensitive single-molecule spectroscopies have been widely used to explore the conformation dynamics of proteins in biological systems – and an ideal probe should yield high signal with low background noise and have good photostability and biocompatibility with a minimum size to avoid interfering with the system of interest,” explained Wei-Shun Chang, a postdoctoral scientist in the department of chemistry at Rice University. He noted that the conventional probes – organic dyes and quantum dots – suffer time-dependent intensity fluctuations (photoblinking) and allow only a limited measurement time due to irreversible photochemical reactions (photobleaching) – and that often they are not biocompatible. “Gold nanoparticles are very photostable and biocompatible, however,” he said. “A surface plasmon of the gold nanoparticle can be photoexcited when the incident light is the same phase and frequency as the collective oscillation of conduction band electrons in metal nanoparticles,” he said. Nanorods vs. spheres “In particular,” he added, “the surface plasmon can be excited with excitation polarization parallel and perpendicular to the long axis of rodlike nanoparticles – nanorods – corresponding to the longitudinal and transverse surface plasmon resonances, respectively. These qualities of gold nanorods make them an ideal vector probe for biological systems.” The graph (left) shows how nanorods photographed in an electron microscope (right) appear and disappear, based on their orientation, when their photothermal signatures are detected with polarized lasers. Photo courtesy of Wei-Shun Chang. Nanorods can be used as vector probes to sense structural changes of proteins in living cells or to detect the local order and fluidity of membranes. Knowing the orientation of the probe can provide important information about the conformation and dynamics of the biological system, according to a report by Chang and his colleagues, which was published in the Feb. 16, 2010, issue of PNAS. Chang works under the supervision of Stephan Link, principal investigator for the project. Link is an assistant professor in the departments of chemistry and electrical and computer engineering at Rice University. Detecting smaller particles “A problem is that single-particle dark-field scattering microscopy cannot detect particles with sizes smaller than 50 nm and, as a result, a new approach was required to detect the orientation of smaller nanoparticles,” Chang said. “In a recent study, our group extended photothermal heterodyne imaging to include polarization-sensitive imaging for detecting the orientation of the nanorods (25 and 75 nm in diameter and length, respectively) by exciting either the longitudinal or transverse surface plasmon resonances.” Particularly, he said, determining the orientation of nanorods by excitation of the transverse plasmon mode cannot be achieved by dark-field microscopy because of the small diameter of 25 nm. “In addition,” he explained, “the resonance maximum of the longitudinal plasmon mode shifts significantly with changes in the nanorod aspect ratio and refractive index of the surrounding environment. The peak shift results in an inefficient excitation of the longitudinal plasmon band with a single-wavelength laser source, and hence a multicolor laser excitation source is required.” “On the other hand, the transverse surface plasmon is insensitive to changes in the aspect ratio and refractive index of the surrounding environment, which means that excitation with a single wavelength is possible, independent of the nanorod aspect ratio,” Chang said. “Due to the high sensitivity of photothermal imaging, the size of the nanorods can be further reduced to below 10 nm, which then becomes comparable to the size of large fluorophores and semiconductor quantum dots. Overall, such ‘small’ nanorods, together with polarization-sensitive photothermal imaging, make this combination an ideal vector probe for applications in biology,” he explained. The setup Photothermal imaging requires a time-modulated heating beam and a probe beam. The scientists used a 514-nm argon-ion laser and a 675-nm diode laser as heating beams to excite the transverse and longitudinal surface plasmon resonances, respectively. The polarization of the heating beam was controlled by a quarter- or a half-wave plate. The heating beam was modulated at 400 kHz by an acousto-optical modulator. Both heating and probe beams were directed into an inverted microscope and focused on the sample through a microscope objective, Chang explained. The signal was collected by the same objective, detected by a photodiode and fed into a lock-in amplifier that was connected to a surface probe microscope controller. Photothermal images were acquired by moving a two-dimensional piezoscanning stage on which the sample was mounted. Alignment of nanomaterials When asked why it was useful to understand how nanomaterials align, Chang answered that scientists and engineers are interested in fabricating different nanostructures. There are two main strategies of fabrication, he said. Top-down strategies employ conventional lithography and etching techniques to fabricate nanostructures from larger bulk materials. In the bottom-up approach, nanostructures are built from smaller units using chemical or physical interactions between the different units, i.e., building blocks. Understanding how these building blocks align one particle at a time will help the scientists to manipulate them and to build more complex nanostructures. “For applications as sensors in biology, the orientation of the nanorod probe could yield the alignment of proteins or measure the order of the local environment, such as in membranes,” Chang said. Measuring absorption and scattering “When photoexciting metallic nano-particles, photons will be either scattered or absorbed by the surface plasmon. The absorbed energy relaxes through nonradiative processes into heat because of a low fluorescence quantum yield,” Chang said. Various metallic nanostructures have been developed for many possible applications, such as subdiffraction-limited waveguiding and surface-enhanced Raman spectroscopy. Nanostructures can also be used as photothermal agents for destroying cancer cells or as contrast agents for bioimaging, he added. To understand the underlying mechanisms for these plasmonic applications, one has to determine both radiative (scattering) and nonradiative (absorption) properties of plasmonic nanoparticles. For example, it is important to measure the pure absorption spectrum of nanoparticles for applications as photothermal cancer agents to ensure an efficient energy conversion from the incident photons to heating the nanoparticle environment. “Alternatively, large scattering cross sections are required for nanoparticles that are used as imaging contrast agents based on plasmon resonance scattering. However, compared to single-particle studies, an ensemble spectrum of metallic nanoparticles yields the overall extinction, which is the sum of scattering and absorption,” Chang said. “Still, photothermal imaging combined with dark-field scattering microscopy for the same nanostructure allows one to determine the absorption and scattering properties separately and as a function of size when the same sample is also imaged by scanning or transmission electron microscopy,” said Chang, who added that the next step will be to incorporate the new technique into a real biological system.