Seeing is believing

A novel method for nanoparticle characterisation allows the direct visualisation of nanoscale particles

Quantum dots emit light in a variey of colours

Nanoparticle characterisation is a key technique for material science research and quality assurance to ensure accurate performance and stability of materials and industrial processes. Accurate information on particle size and shape is also critical in the pharmaceutical industry to detect contaminants or aggregates for process optimisation and control. In addition, pharmaceutical and biotechnology research represents an exciting and novel application to characterise nanoparticles for drug delivery and enhanced performance.
For example liposomes continue to grow in importance as a method for drug delivery as they protect the drug from the actions of metabolising enzymes and can be targeted to specific areas in the body; lowering dose rates, thereby reducing toxicity and side-effects. Liposome size is has been recognised as an important factor in treatment efficacy and efficiency as liposome size may affect its circulation and residence time in the blood, the efficacy of targeting, the rate of cell absorption (or endocytosis) and, ultimately, the successful release of its payload. Accurate measurements of nanoparticle size, concentration and size distribution of liposomes prior to administration serve as an important tool for treatment efficacy.
With the increasing demand for nanoparticle characterisation, new systems are required that increase the accessibility, flexibility and sensitivity of traditional techniques such as Photon Correlation Spectroscopy (PCS).
Photon Correlation Spectroscopy is a powerful method that has been used to study the dynamics of particles in suspension. It collectively analyses a large ensemble of particles from which only an intensity weighted (z-average) particle mean is obtained as well as a polydispersity quotient indicating the width of the particle size distribution2. As a large number of particles are measured simultaneously, in PCS, it is frequently the case that a relatively small number of highly scattering larger particles such as contaminants and/or aggregates can effectively dominate the signal generated by the far larger population of smaller particles that may be present. It is possible, however, through the application of various de-convolution algorithms, to extract particle size distribution structure from the results obtained but this approach is reliable only if the two populations are not too polydisperse themselves or too close together in size.

NanoSight LM10 sample chamber

New systems have been introduced to avoid problems associated with traditional nanoparticle characterisation systems. The Nanosight LM10, is a novel method for nanoparticle characterisation that enables real time visualisation and sizing of individual nanoscale particles in suspension. Given that each and every visible particle is separately tracked, it is possible to generate particle size distribution profiles that reflect the actual number of particles; a significant advance on those distributions obtained by PCS. Uniquely, this technique enables the direct visualisation of nanoparticles in suspension in real time. The new technology, used on its own or to complement other techniques, is expected to make a significant impact on nanoscience across a wide range of application areas. The upper limit of 600nm for this system represents the bottom of the size range for which other particle sizing techniques can be used, such as diffraction techniques or microscopy.
The Nanosight LM10 system is available as a complete microscope based system using only a conventional optical microscope fitted with a specialised camera and dedicated analytical software1. Nanoparticles suspended in a liquid are illuminated by a laser beam and can be seen through a conventional optical microscope as small points of light moving rapidly under Brownian motion. The system can analyse any nanomaterial down to 10-30nm diameter, dependent on the nature of the particle, with minimal pre-treatment: only pre-filtering to remove large contaminants/aggregates and dilution with a suitable solvent to an acceptable concentration range.
Specialised Nanoparticle Tracking Analysis (NTA) software, also developed by NanoSight, analyses particle movement from the video sequence and determines the mean squared displacement per unit time for each particle for as long as it is visible. From these values, the diffusion coefficient and hence sphere-equivalent, hydrodynamic radius can be determined using the Stokes-Einstein equation. The close to real-time nature of the technique enables accurate determination of particle-particle interactions through information about aggregation and dissemination within a sample. The accuracy of results obtained by the Nanosight LM10 system is dependent on a number of factors including particle concentration analysed, the length of analysis time and the size of the particles present. Sample concentrations between 106 and 1010 particles/ml are necessary for a statistically significant number of particles to be present on the beam. Larger sized particles become increasingly difficult to size accurately however this can be improved by extending analysis times or image smoothing.
PCS has a very limited ability to produce accurate particle size distributions from a poly-dispersed sample. The signal generated by PCS for the larger particles effectively obscures that of the smaller particles, leading to loss of data relating to the smaller particle peak. Only when the level of light scattered from each particle population is matched, each has a narrow size distribution and the particle populations are sufficiently dissimilar in diameter (e.g. >2:1), will PCS be able to easily produce accurate bimodal distributions. Due to the unique ability to see and subsequently ascribe particle size on an individual particle basis, the Nanosight LM10 system does not suffer from these limitations.
Figure 1 shows the benefit of being able to analyse particles individually for size by Brownian motion without having to rely on an intensity weighted average of a large ensemble of particles as is the case PCS. In this case a suspension of 200nm particles was mixed with a lower number of 400nm particles (Figure 1a) and analysed for 20seconds by the NanoSight NTA system (Figure 1b) and conventionally by PCS ((Malvern Instruments 4700CE  - Figure 1c). The results of the PCS analysis (Figure 1c) show that  the signal generated in PCS by the larger particles effectively obscures that generated by the smaller particles leading to loss of data relating to the smaller particle peak.
This is in significant contrast to the results obtained by the NanoSight NTA system in which both peaks are clearly visible (Figure 1b). Figure1d shows the same profile displayed on a log scale for a direct comparison to the PCS data. The unique ability to visually confirm the presence of more than one particle population within a sample has proved to be extremely important in being able to validate the data obtained by other techniques such as PCS in which the user might otherwise be “working blind”.
Finally, it should be noted that the Nanosight LM10 system allows the number of particles of any given size that were seen during the analysis to be counted directly representing an advantage over conventional PCS which cannot generate such information.
A simple and direct qualitative view of the sample under analysis, using the Nanosight LM10, enables highly accurate and sensitive nanoparticle detection. The video sequence is used to immediately determine independent quantitative estimation of sample size, size distribution and concentration at a tenth of the cost of electron microscopy techniques traditionally used for this purpose. The system can measure nanoparticles down to 10nm, dependent on material, and can obtain higher resolution particle size distribution profiles than other more time-consuming and expensive methods. Its ability to directly visualise and ascribe particle size on an individual basis without relying on an intensity weighted average, as in PCS, enables separate populations to be better resolved in polydisperse systems.
The direct and close to real-time nature of the Nanosight LM10 nanoparticle characterisation system means that it is rapidly being considered a key technology for the routine detection and monitoring of a wide range of virus types. The LM10 can detect and analyse enveloped viruses, such as influenza, despite their lack of high refractive index capsid coat material. Its relatively low cost and ease of operation means that the system is a widely accessible method for the detection of viruses which may have important implications for the monitoring of biohazards and viral epidemics, such as Avian Flu.
The LM10 system is being continually developed to support the growing research needs of customers across the chemical and biotechnology industries. The development of new software algorithms to enhance its sensitivity and accuracy as well as extension of new techniques, such as measurement of zeta potential, will support customers growing analysis requirements.

By Dr Bob Carr.
Dr Carr is the CTO of NanoSight Ltd. and started working on nanotechnology whilst at CAMR. Bob has expertise in the areas of microbiology, optoelectronics and microfluidics.