Andrew Malloy from NanoSight tells Laboratory News about new instruments to track nanoparticles
Nanoparticle Tracking Analysis (NTA) can visualise, count and size nanoscale particles such as viruses and virus aggregates in real-time. It simultaneously and directly measures the diffusion coefficient of each virus in solution, and from this calculates high-resolution particle size versus concentration distributions which are key in evaluating both viral titre and state of aggregation. With minimal sample preparation, this technique provides unprecedented and quantified insight into a viral preparation. The unique data generated by the technique is also backed up by a qualitative image of the sample where the user can instantly recognise key characteristics in the preparation.
Instruments from NanoSight use a laser light source to illuminate virus and other nanoscale particles. Enhanced by a near-perfect black background, the particles appear individually as point-scatterers moving under Brownian motion, Figure 1. Purified versus freshly harvested viral suspensions are instantly distinguishable and quantifiable.
Fluorescence mode may be used to discriminate populations under scrutiny from non-labelled backgrounds. Three wavelengths may be used to excite fluorescence (405nm, 470nm and 532nm) and the fluorescent emissions may also be imaged from particles which have been suitably labelled.
Titre results are available by particle size class and are displayed as frequency size distributions and output to a spreadsheet. Supplementary scatter intensity data is combined with these size distributions to provide 3D plots which highlight disparate populations. Video clips of images are recorded and retained for further analysis, figure 2.
The NTA technique provides size and count of all the particles present whether live or inactive. This allows the user to quantify the relative concentrations of infective particles versus total particles when used in conjunction with infectivity assays. The ability to monitor the ratio of infective versus non-infective viruses is key in process development and provides vital information in understanding where losses in the purification process occur (both in terms of absolute number of viruses and also state of aggregation).
Figure 1 Schematic to illustrate how nanoparticles are visualised and their images captured
Used alongside quantitative real-time polymerase chain reaction (qPCR), NTA can be used to quantify the number of filled versus empty capsids – whereas qPCR can only measure viruses with DNA/RNA, NTA makes no differentiation between filled and empty capsids (unless operating under fluorescence mode in which the viral DNA/RNA has been appropriately labelled).
For Virus-Like Particles (VLPs) which contain no DNA (i.e. they are non-infectious), neither infectivity assay nor qPCR can be used. As such, NTA – which requires neither infectivity nor DNA/RNA to provide size and concentration – represents an ideal tool for their characterisation.
NTA is unique in that it provides a direct, real-time live view of all particles in suspension. It is able to complete the measurement of total particle titre in approximately one minute meaning it can differentiate between infectious and non-infectious viruses.
Being able to measure particle-by-particle with such high resolution sizing enables the differentiation of single virions from their aggregates. The reported data also provides scattering intensity distribution as well as evidence of non-sphericity and of particle aspect ratio.
The minimum detectable size depends on particle type but most virus and phage types can be seen, sized and counted. By having the ability to make a real-time measurement of size and concentration, aggregation kinetics may be readily studied. The effect of changing temperature, pH or excipients may also be quantified in real-time.
Figure 2 Image showing 100nm Virus-Like Particles with their trajectories shown, from which their size can be determine
The strength of NTA shows well in the study of polydisperse samples where there is clear differentiation even when particles of similar size are observed. There is also no bias in the results towards particles of larger size which is clearly apparent in other light scattering techniques such as dynamic light scattering (DLS).
What makes these applications so straightforward to perform is the instrumentation providing ease of use along with accurate, reproducible data in a reliable system package. The latest member of NanoSight’s family is the NS500 which provides many new capabilities developed from interactions with users from all over the world. A high sensitivity EMCCD camera is now standard to enable fluorescent measurements to be routine. A choice of red, green and blue lasers may be used to suit specific applications.
The addition of fluid handling to the NS500 provides the user with auto sample presentation and in-situ cleaning. It is now a routine process to clean the cell with the ability to purge, flush and load samples through user-customisable software. Dilution may also be optimised then controlled in this way. Ease of use is further enhanced with a motorised focus function to readily find the particles under study. In direct response to user feedback, this is augmented with an indexed motorised stage, controlled through the software and providing excellent repeatability in positioning. The temperature control of the cell offers a broad range (15°C to 55°C) and programmable temperature cycling, with rapid attainment of set-point facilitating faster sample measurement and turnaround, vital for the study of kinetic events such as protein aggregation.
With NTA, there are many aspects of virus characterisation which may be studied now it is possible to handle measurements on the 10-1000nm range so easily.
When used in combination with infectivity assays, NTA can be used to calculate total versus infective viruses. This is critical in process development as this ratio often exceeds 100:1 meaning that 99% or more of the total viral population is inactive and that the technique opens the way for better process development. The method may be applied at all stages of production from the initial harvest through to the downstream purification steps and packing.
The measurement of aggregation is important as this can be indicative of the long term storage stability of the product. Being such a quick technique to use, NTA may be used to check the efficiency of filters used in the purification process by monitoring the purity of the viral spikes. The degree of aggregation and the accurate quantification of the total viruses present will provide an indication of the ability to filter the virus.
Fluorescence is used to provide differentiation between labelled particles from the complex background which gives qualitative data in advance of the purification process.
The efficiency of the delivery vector for gene delivery applications will be indicated from the measurement of viral preparations. This will be observed from changes in concentration, purity and stability of the product.
Phage therapeutic studies benefits from the easy measurement of the purity and concentration of bacteriophage preparations.
The flexibility of the technique has been demonstrated on many different viruses to date. It is also of note that the technique has achieved excellent acceptance worldwide with more than one hundred third party peer-reviewed papers citing the use of NTA.
The ability of the NanoSight instrument to count and size viruses and their aggregates in liquid suspension is becoming increasingly important to those involved in the development of viral vaccines. For more examples of this type of study and to look at examples of videos of virus particles being captured, readers are recommended to visit the NanoSight web site: www.nanosight.com.