Fundamental Questions

Size range

What is the largest size of particles that can be analysed?

The upper size limit is approximately 1000 nm. The NanoSight NTA technique analyses the movement of a particle undergoing Brownian motion by tracking the spot of light it scatters. For large particles:
1. The diffusion of the particle reduces making the accurate determination of its mean displacement progressively less accurate.
2. For dense particles, sedimentation rates become significant compared to the limited Brownian motion.
3. The spot of light becomes so large and its edges variable between frames, thus identifying its centre becomes increasingly difficult, leading to centring errors which are as large as the particle's movement.

These effects get progressively larger as particle size increases so there is no specific limit to our upper size range. This upper limit will depend on the nature of the system. In practice, for low density and low Ri particles such as polystyrene micro-spheres their buoyancy and weaker scattering allow them to remain Brownian movers with easily identifiable centres and the upper size limit is 1000 nm or above.

For dense, high Ri particles such as gold, their rapid sedimentation rate and excessive scattering restricts the upper size at which they can be analysed to approx <200 nm.

What is the smallest particle that can be analysed?

The minimum size of particle that is detectable depends on the scattering being sufficiently strong. To understand this question better see 'How much light is scattered by a particle?'. As a general guide:
1. Low Ri (e.g. biological/polymer) will be detectable down to approximately 40-50 nm.
2. Higher refractive particles (e.g. ceramics, metal oxides) will be detectable down to approximately 10-25 nm.
3. Exceptionally high Ri particles (e.g. Ag sol, Au colloid) can be detected at diameters as low as approximately 10 nm.

How does a larger particle (such as an advanced agglomerate or contaminant) affect the measurement?

Unlike other light scattering techniques we are not strongly affected by large contaminants. This is because we visualise individual particles. Whilst an unusually large particle will scatter far more than the primary particles, its scattered light will be confined to and tracked in a region of the image.

Light scattering

How much light is scattered by a particle?

The particles that we size are generally in the Rayleigh or Rayleigh-Debye-Gans scattering regime. As an approximation briefly the Rayleigh regime will be discussed. In this case the efficiency of light scattering is given by:

Therefore the factors affecting the amount of light scattered are:
d = the particle diameter. If you double the particle size you increase the power of light scattered by a factor of 64.
λ = the wavelength of light. If you half the wavelength of light you increase the scattering by a factor of 16.
n = the ratio of the particle refractive index to the solvent refractive index.

For example for latex particles (ref. ind. 1.59) in water (ref. ind. 1.33), n is 1.195 and the factor  is 0.0157.

However for the case of gold (ref. ind. 0.2-3.37i) in water n is 0.15+ 2.53i and  is 2.78, a factor of 177 greater than for the latex particles.

The amount of light scattered is also directly proportional to the intensity of incident illumination.

2D observation

The particles are only being imaged in two dimensions, but they must be moving in three dimensions. Is this a problem?

No, the Stokes-Einstein theory that is used to calculate the particles size from the particle motion can take into account whether two or three dimensions are tracked (see equations below).

Concentration

What is the best particle concentration to use?

Between 10⁷-10⁹ particles per ml.

Twinkle effect

Why do my particles twinkle and appear to diffuse asymmetrically?

Whilst currently we cannot quantitatively answer this, our understanding is that this is due to the particles being oblate (non spherical).