Biomaterial characterization at the nanoscale is gathering interest, as a better understanding of properties such as particle size or number may provide new ways to look at nanotoxicology or provide pathways to the development of early diagnostic test protocols. Extracellular microvesicles and exosomes (nanovesicles) are emerging as a significant class of submicron structures of potentially great importance in the diagnosis of a wide range of disease states and the development of treatment. Found in all organisms and generated by nearly all cells, they are believed to contain a variety of signaling proteins as well as genetic material of many different types. To date, detection of these particles has been possible only through electron microscopy or by classical methods of analysis such as dynamic light scattering (DLS). Flow cytometry has a lower limit of some 300 nm and therefore cannot see the majority of microvesicular material thought to be present. A new technique, nanoparticle tracking analysis (NTA), allows exosomes and microvesicles in the size range of 30 to 1000 nm in liquid suspension to be directly and individually visualized in real time, and it provides high-resolution particle-size distribution profiles and concentration measurements. The technique is fast, robust, accurate and cost-effective, representing an attractive alternative or complement to existing methods. Specific exosomes can be imaged and characterized through suitable labeling with a fluorescent marker, and a range of excitation wavelengths is available. How NTA works The instrumental design, patented by NanoSight Ltd. of Salisbury, UK, uses a laser light source to illuminate nanoscale particles in liquid. Enhanced by a near-perfect black background, the particles appear individually as point-scatters moving under Brownian motion (Figure 1). Polydisperse and multimodal systems are instantly recognizable and quantifiable. Figure 1. A schematic of the NanoSight NS500 nanoparticle tracking analysis system. Using proprietary image analysis software, NTA automatically tracks and sizes particles simultaneously in real time. Results are displayed as frequency-size distributions or in a variety of user-chosen variables. The measurement process requires three simple steps. Samples are prepared in an appropriate liquid (typically water-based) at a concentration level of 107 to 109 parts per milliliter (ml) and placed into the sample chamber, which has a volume of 3 ml. The laser then illuminates the samples in the chamber, and the dispersed light is captured via the microscope by a high-sensitivity camera. Particles are individually tracked and visualized on the screen of the control computer. The smallest appear as fast-moving dots of light; larger particles move more slowly and show more scatter. A “movie” of the moving particles is captured and stored at a rate of 30 fps; this data shows the individual “track” of each particle. The user selects the final output, from a simple particle distribution curve to a complex 3-D display, enabling different materials of very similar particle size to be easily differentiated. This process is illustrated in Figures 2a and 2c. Figure 2. (a) A still image of nanoparticle suspension as seen by a microscope in the path of the laser beam; (b) trajectories of an individual particle’s Brownian motion as plotted by the tracking analysis program; and (c) a particle-size distribution profile as generated by analysis of particle trajectories. Direct observation of particle motion and scattering behavior provides a wealth of information beyond particle size. These real-time observations validate the reported particle-size distributions and provide instant insight into polydispersity and state of aggregation. Measurements take just minutes, allowing time-based changes and aggregation kinetics to be quantified. Although both follow the dispersed light from the particles through their Brownian motion, NTA differs significantly from DLS, also known as photon-correlation spectroscopy: DLS measures change in scatter intensity of the bulk sample, but NTA measures observed particle diffusion directly, particle by particle. The NTA method measures the diffusion coefficients of individual particles and builds the distribution one particle at a time. This compares favorably with an ensemble measurement of the combined light scattering intensity of a population of particles. Consequently, rather than presenting an ideal curve driven by a range of assumptions, the results from NTA are a true high-resolution particle-size distribution; therefore, this approach is especially strong for characterizing complex poly-disperse systems. Instrumentation As NTA has evolved, diverse applications have required various lasers for illumination supported by more sensitive cameras. Fluorescence labeling also has been used in many applications, particularly in the life sciences. In all cases, the instrumentation must provide ease of use along with accurate, reproducible data in a reliable package. System design has focused on providing rapid solutions in both research and industrial environments: A high-sensitivity electron-multiplying CCD camera enables fluorescence measurements with a choice of red, green and blue lasers to suit specific applications. Fluid handling provides the user with automatic sample presentation and in situ cleaning. With user-customizable software, the cell can be cleaned and samples can be purged, flushed and loaded. Dilution may also be optimized, then computer-controlled in this way. Figure 3. The NanoSight NS500 nanoparticle characterization system was developed with input from users worldwide. Its sensitive electron-multiplying CCD camera enables fluorescence measurements in a variety of wavelengths for specific applications. A motorized focus function readily finds the particles under study. In response to user requests, this is augmented by an indexed motorized stage, controlled through the software and providing excellent repeatability in positioning. The temperature control of the cell offers a broad range (15 to 55 °C) and programmable temperature cycling. Rapid attainment of set point ( Applications: exosomes, microvesicles Not only does NTA offer a way to see and count extracellular microvesicles and exosomes, but variations in the technique – a fluorescence mode, for example – allow exosomes to be phenotyped as well. This multiparameter capability, compatible with natural structures in their native environment, promises to help elucidate the roles these structures play in disease and the ways in which they can be exploited in a diagnostic or therapeutic application. Both flow cytometry and NTA have shown extremely encouraging results in the rapid sizing, quantitating and phenotyping of cellular vesicles, according to data reported by a group of researchers from the University of Oxford in the UK, led by professor Ian Sargent.1,2 Their interest was in the study of cellular microvesicles (100 nm to 1 µm) and nanovesicles ( Protein aggregation NTA has been proposed, used and assessed in the study of protein aggregation and in the characterization of virus preparations and viral vaccine products. The presence of previously subvisible particles in therapeutic protein products may have a detrimental effect on product quality, noted professor John Carpenter of the University of Colorado at Denver.3 This has been borne out in subsequent papers, such as one by F. Gruia,4 in which he described the characterization of submicron particle distributions in biologics formulations and suggested that NTA is a novel technique with the potential to enhance the current analytical capabilities for detecting, sizing and counting particles in the submicron range. Figure 4. Size-distribution measurements from nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) measurements of mixtures of monodisperse polystyrene beads (middle panels) with the corresponding NTA video frame (left panels) and 3-D graph (right panels: size vs. intensity vs. concentration). (a) 60-/100-nm beads at a 4:1 number ratio; (b) 100-/200-nm beads at a 1:1 number ratio; (c) 200-/400-nm beads at a 2:1 number ratio; (d) 400-/1000-nm beads at a 1:1 number ratio.4 These findings complement studies done at the University of Leiden by professor Wim Jiskoot and his colleagues. His colleague Vasco Filipe led a critical evaluation of NTA for measuring nanoparticles and protein aggregates.5,6 He found that NTA accurately analyzes the size distribution of monodisperse and polydisperse samples, and that the sample visualization and individual-particle tracking features enable a thorough size distribution analysis. They confirmed that the presence of small amounts of large particles (1000 nm) generally does not compromise the accuracy of NTA measurements, and that a broad range of population ratios could easily be detected and accurately sized. NTA was shown to be suitable for characterizing drug delivery nanoparticles and protein aggregates. The investigators concluded that NTA complements DLS and is particularly valuable for analyzing polydisperse nanosize particles and protein aggregates. Nanotoxicology While the rapid development of a wide range of materials and products containing nanoscale structures and engineered nanoparticles continues, awareness has grown that the longer-term potential toxic effects of such materials and their environmental impact are poorly understood. Parameters such as particle size, number and state of aggregation are extremely important. Nanoparticle measurements are being performed in a wide range of locales, including human cells and wastewater. Consumer products such as sunscreen and toothpaste use nanoparticles as active ingredients. In sunscreen, it is titanium dioxide that provides reflective protection,7 while in toothpaste, nanosize components play a role in the abrasive cleaning processes.8 The multiparameter analysis of nanoparticles using NTA is already well documented with more than 350 peer-reviewed papers in press since 2006. This is an incredibly large number for a technique still in its infancy – where new applications are discovered week by week. More importantly, the ability to provide quantitative data on materials that are not well understood will be invaluable to the world in general. Meet the authors Bob Carr is the CTO and founder of NanoSight Ltd. of Salisbury, UK. Patrick Hole is NanoSight’s head of development. Andrew Malloy is head of sales; email: andrew.malloy@nanosight.com. References 1. R.A. Dragovic et al (December 2011). Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine: Nanotechnology, Biology and Medicine, pp. 780-788. 2. R.A. Dragovic et al (July 2011). Development of flow cytometry and fluorescence nanoparticle tracking analysis (NTA) to characterise cellular microvesicles and nanovesicles. Flowcytometry UK, Royal York Hotel, York, UK. 3. J.F. Carpenter et al (April 2009). Overlooking subvisible particles in therapeutic protein products: Gaps that may compromise product quality. Journal of Pharmaceutical Sciences, pp. 1201-1205. 4. F. Gruia (2012). Characterization of submicron particle distributions in biologics formulations. PEPTalk, January 9-13, San Diego. 5. V. Filipe et al (May 2010). Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharmaceutical Research, pp. 796-810. 6. V. Filipe et al (May 2011). Fluorescence single particle tracking for the characterization of submicron protein aggregates in biological fluids and complex formulations. Pharmaceutical Research, pp. 1112-1120. 7. A.C. Johnson et al (June 2011). An assessment of the fate, behaviour and environmental risk associated with sunscreen TiO2 nanoparticles in UK field scenarios. Science of the Total Environment, pp. 2503-2510. 8. A. Peetsch and M. Epple (February 2011). Characterization of the solid components of three desensitizing toothpastes and a mouth wash. Materialwissenschaft und Werkstofftechnik, pp. 131-135. Acknowledgment NanoSight thanks the authors and the publisher for permission to reproduce the set of images in Figure 4. The journal citation is V. Felipe et al (See Ref. #5.), doi: 10.1007/s11095-010-0073-2 (open access at Springerlink.com).