Nanoparticle Tracking Analysis Particle analysis using a microscope

Nanoparticle tracking analysis (NTA) particle measurement technology examines the speed of motion of single particles in a suspension using the models of Brownian motion and light scattering. It determines particle sizes, particle size distributions as well as particle concentrations. In this article we describe what nanoparticle tracking analysis is, how it works, what it is used for and where our patented OF2i® method is a better alternative.

Table of contents

What is nanoparticle tracking analysis?

Nanoparticle tracking analysis or NTA is a technique used for the characterization of nanoparticles dissolved in a liquid sample. The NTA method is well-suited as an analytical measuring procedure to determine particle sizes, particle size distributions, particle concentrations as well as the zeta potential. It is even possible to monitor single particles. The nanoparticle tracking analysis method makes use of the random movement of particles (Brownian motion) as well as the scattering of light to calculate the diameter of particles with the aid of the Stokes-Einstein equation and the diffusion coefficient. The particle size range for measurement is between 10 nm and 20 µm. This means NTA can also be used to measure aggregates. The sample is measured in a dispersion medium, like water or an organic solvent, in which a laser beam is used to monitor the speed of movement of the particles. The structure of a nanoparticle tracking analysis instrument allows monitoring of the particle behavior in real-time, although only for a very small sample amount.

How does nanoparticle tracking analysis work?

The basic structure of an NTA instrument consists of five components. The sample (in suspension) is measured in a cell chamber which is illuminated by a laser. The measurement results are captured by a microscope and a CCD or CMOS camera and a computer evaluates and prepares the data. Nanoparticle tracking analysis uses Brownian motion and scattered light to characterize nanoparticles. This works by directing a laser beam through the cell chamber in order to illuminate the particles in the liquid from the side. The laser light is scattered whenever it hits a (nano)particle. The scattered light is captured by the microscope and recorded by the camera, which is directly installed on the microscope. In this way NTA can measure the Brownian motion of all the particles inside the laser beam and determine the diffusion constant. By using the Stokes-Einstein-equation the hydrodynamic diameter of single particles can be calculated and the particle size, particle size distribution and particle concentration are calculated. Besides this, nanoparticle tracking analysis can measure the electrophoretic mobility and the zeta potential of the sample.

Brave Analytics Nano Particle Tracking Analysis Illustration of strucutre

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What can be measured by nanoparticle tracking analysis?

With nanoparticle tracking analysis you can measure the particle size, particle size distribution and the electrokinetic potential (zeta potential). It is also possible to measure the particle concentration although this value has relatively low accuracy. NTA calculates the diffusion parameters and the hydrodynamic diameter of the particles. Some nanoparticle tracking analysis instruments have a fluorescence mode which allows fluorescent tagging of certain particles of a sample. With nanoparticle tracking analysis, all of these parameters can be measured simultaneously. Even changes in the characteristics of the particle populations can be measured over time, although the results are not representative of the whole particle population as NTA  only analyzes a small number of particles per minute. By measuring the subpopulations NTA provides insights into the heterogeneity of very complex samples. Furthermore, a quantification of colocalization relations is possible.

The nanoparticle tracking analysis principle

Just like the dynamic light scattering method, nanoparticle tracking analysis uses the models of the random molecular motion (Brownian motion) and the scattering of light to determine particle sizes and particle size distributions. Brownian motion describes the random movement of nanoparticles that are suspended in liquids (e.g. in water or organic solvents like ethanol). These movements are also called diffusion and expressed by the diffusion coefficient (D). Brownian motion is triggered by the energy transfer from the molecules of the liquid to the particles in the sample. Different formulas are used to determine the diffusion coefficient depending on the number of dimensions. By using the Stokes-Einstein equation, the particle diameter can be calculated (as a function of the diffusion coefficient at a certain temperature and a certain viscosity of the liquid). Finally, the nanoparticle tracking analysis principle combines the Stokes-Einstein equation with the two-dimensional mean square deviation to calculate the fluctuations of a single particle and its diameter.

The advantages of nanoparticle tracking analysis

Unlike ensemble light-scattering methods like DLS, the scattering of large particles does not affect the results of nanoparticle tracking analysis as it measures with single-particle sensitivity, at least if the particle concentrations lie within the particle size range. Thus NTA works well for the analysis of nanoparticles of greatly varying size distributions. One benefit of NTA is the possibility to monitor processes like agglomeration continuously, although the observed sample  amount is limited and therefore not representative. Measurement results are delivered in a matter of minutes and sample recovery is also possible.

The advantages of NTA in a nutshell

The limits and disadvantages of nanoparticle tracking analysis

Although NTA is designed to analyze low-concentrated samples, it does not provide useful measurement results for ultra-low particle concentrations. Representative measurement results are obtainable if the particle concentration lies between 106 and 108 particles per milliliter. However, with NTA it is not possible to obtain truly representative measurement results for the whole particle population as only approximately 100 particles per minute can be measured per frame. As the volume of the sample in the cell is dependent on the particle sizes in the sample, the accuracy of the calculated particle concentration is relatively low. A further drawback when using NTA is the time-consuming manual cleaning of the measuring chamber .

The disadvantages of nanoparticle tracking analysis in a nutshell

What is nanoparticle tracking analysis used for?

Nanoparticle tracking analysis is used to characterize APIs such as liposomes, polymeric nanoparticles or virus-like particles, to detect aggregates inside of protein compositions or detect viral particles as well as to measure non-organic particles and bio-nanoparticles. However, this only works if the particles are big enough. If this is not the case, the patented OF2i method is a good alternative. For the measurement of polydisperse samples, nanoparticle tracking analysis is a better choice than DLS, although protein monomers typically prove too small for NTA measurements. OF2i can measure protein monomers and provides highly representative data for polydisperse samples. Furthermore, nanoparticle tracking analysis is used for stability studies to monitor changes in particle concentrations. It is also used for the analysis of abrasives, polishing agents, coatings, inks, filler materials, ceramic, pigments, food and beverages. Samples suited for analysis by NTA include:

Five reasons why OF2i® is a better alternative to nanoparticle tracking analysis


Nanoparticle tracking analysis cannot measure ultra-low particle concentrations whereas OF2i® measures concentrations of only a few particles per milliliter.


As nanoparticle tracking analysis measures approximately 100 particles per minute/frame the results are not representative of the whole particle population, OF2i® measures up to 1000 particles per minute and delivers statistically relevant results, even for polydisperse samples.


OF2i® delivers much faster results, depending on the sample it is between three and ten times faster than NTA.


OF2i® allows seamless monitoring of particle behavior in real-time and for seconds, minutes or hours.


OF2i® can be integrated into your production plant to monitor and control processes (PAT sensor).


ASTM E2834-12, Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Nanoparticle Tracking Analysis (NTA), ASTM International, West Conshohocken, PA, 2012,

Bohren CF, Huffman DR. Absorption and scattering by a sphere. In: Bohren CF, Huffman DR, editors. Absorption and scattering of light by small particles. Weinheim: Wiley-VCH Verlag GmbH; 2007. pp. 83–129.

Brown, Robert (1828). “A brief account of microscopical observations made in the months of June, July and August, 1827, on the particles contained in the pollen of plants; and on the general existence of active molecules in organic and inorganic bodies” (PDF). Philosophical Magazine. 4 (21): 161–173. doi:10.1080/14786442808674769

Einstein, A., Annalen der Physik, 19 (1906), Seiten 371-381, Zur Theorie der Brownschen Bewegung

Giebel B, Helmbrecht C. Methods to Analyze EVs. Methods Mol Biol. 2017;1545:1-20. doi: 10.1007/978-1-4939-6728-5_1. PMID: 27943203.
Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Qian, H., Sheets, M. P., Elson, E. L., Single particle tracking, Biophys J. 1991 Oct; 60(4): 910–921. doi: 10.1016/S0006-3495(91)82125-7

Smoluchowski, von, M., Annalen der Physik, 14, Seiten 756-780, 1906, Zur kinetischen Theorie der Brownschen Molekularbewegung und der Suspensionen

Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility

Vasco Filipe, Andrea Hawe and Wim Jiskoot (2010). “Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates”. Pharmaceutical Research. 27 (5): 796–810. doi:10.1007/s11095-010-0073-2. PMC 2852530. PMID 20204471.

Zsigmondy, R., Colloids and the Ultra Microscope, J. Am. Chem. Soc., 1909, 31 (8), pp 951–952. doi: 10.1021/ja01938a017