Obtaining a good image of the particles is a key step when making a NanoSight measurement. However, the image settings chosen by a user has an influence on the data reported. This has raised concerns from new and infrequent users who worry if the particles are bright enough and in focus, and for experienced users who want to be sure that run-to-run and day-to-day data comparisons are valid.
The Auto Setup function, introduced in the NanoSight NTA 3.1 Software Package, is designed to automatically optimise the imaging parameters for video capture. The image is automatically focussed image and an optimum camera level for measurement is set. This improves reproducibility, by removing susceptibility to any user dependent factors, and for most sample types, improves the repeatability of image optimisation compared to a user adjusting manually. Auto Setup can be used with the NTA Standard Measurement SOP for fully automated image capture settings.
The Auto Focus function for NS300 and NS500 instruments, varies the image focus and sets the optimum focus for NTA image capture. The Auto Focus repeatability performance is specified for non-polydisperse latex microspheres from 50 – 400nm (HS systems), or 100 – 400nm (non-HS systems), within a concentration range of 2x108 up to 1x109 particles/ml. Performance may vary according to specific system configuration, particle refractive index and presence of aggregates.
Auto Focus can be used to set the image focus before each measurement SOP, or run periodically during long-term sample monitoring measurements. For samples which do not meet the criteria above, standard particles can be loaded and used for auto-focussing, immediately prior to loading and measuring the sample.
Auto Focus performance tests show reduced variation compared to the repeatability and reproducibility inherent with different users manually setting the image focus, and typically has better repeatability than a single expert user can achieve manually (Figure 1).
Figure 1: Normal distribution fits showing repeatability of manual focus with two users (red and green curves) and autofocus (blue curve) for 200nm latex microspheres.
Auto Camera Level
The Auto Camera Level feature can be applied to all samples, including polydisperse mixtures on all NanoSight systems. It automatically runs after Auto Focus on NS300 and NS500 instruments. It can also be run independently for systems without motorised focus control, or for polydisperse and aggregated samples unsuitable for auto-focussing. The automatically set camera level provides well resolved size distributions for mixtures of latex microspheres and maintains NTA sizing results within the normal measurement error on NIST size standards.
The camera level is set as high as possible whilst maintaining sizing and concentration calibration accuracy. This allows dimmer particles within a polydisperse population to also be visualised, allowing a broad range of particle sizes to be measured using a single video capture.Auto Camera Level performance tests show resolved particle distributions for mixtures of latex standard microspheres, 100nm+300nm and 100nm+200nm+400nm (Figure 2).
Figure 2: NTA results and video image for a mixture of 100nm, 200nm and 400nm latex microspheres measured using Auto Camera Level.