The Role of Scatter Intensity in NTA Size Measurements

Nanoparticle Tracking Analysis (NTA) determines particle size through diffusion, but particles are not directly visible under the optical microscope. Instead, they are observed via the light scattered from their surface. The intensity of this scattered light is highly sensitive to particle size, as it varies with the sixth power of the diameter. However, many factors can affect scattering intensity, making it challenging to use this information quantitatively. One major cause of variability in previous NTA instruments is the inhomogeneity and lack of reproducibility in the optical system.

Quantitative Intensity Readings rather than Qualitative

The Envision NTA particle analyzer from Hyperion Analytical was designed with a precise optical system to provide quantitative, rather than qualitative, intensity readings. Measurements from various samples demonstrate a clear, positive correlation between particle size and intensity, showing an increase in intensity as particle size grows.

Positive Correlation Between Particle Size and Intensity

This study introduces absolute intensity measurements for scatter and fluorescence in biological research. Samples ranging from 40 nm to 200 nm were tested using the Envision NTA analyzer, a precise optical system for quantitative intensity readings. Two sample sets were tested: the first included 44 nm, 102 nm, and 194 nm latex samples analyzed in both scatter and fluorescence modes, while the second consisted of various polymer samples analyzed only in scatter mode ranging from 73 nm to 122 nm. Consistent camera settings were maintained across all samples to ensure accurate intensity data collection.

Conclusion: Enhancing NTA with Reliable Intensity Data

The positive correlation between particle size and measured intensity is clearly illustrated in Figure 1 and Figure 2, showing an increase in intensity as particle size grows. Figure 2 specifically highlights that fluorescence intensity is lower than scattering intensity due to fewer fluorescence labels on each particle, yet it remains consistent with expected trends. The study shows that intensity can be measured in both fluorescence and scattering modes despite variables like laser power and camera exposure. This emphasizes the need for further testing, especially in fluorescence, given its growing relevance. The ability to measure intensity quantitatively helps researchers track, analyze, and select particle positions, enabling more advanced experiments and investigations, particularly in studying Extracellular Vesicles (EVs).

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