Biomedical Engineering Reference
In-Depth Information
The methods for the measurement of size can be divided into single NM methods, such as SEM,
TEM, and AFM, and ensemble methods, such as dynamic light scattering (DLS) [11]. The advanced
single NM methods can, simultaneously, visualize and more accurately measure the size of the
original NMs if measurement parameters are more optimal than other methods [13]. However,
for polydisperse NMs, statistical significance is a problem since the number of individual NMs in
an acquired high-resolution image is very limited. This issue can be addressed by acquiring mul-
tiple images and using automated image recognition software for the determination of size. DLS
is a conventional method for measuring the size distribution in a suspension. DLS measures the
Brownian motion of NPs and relates this movement to an equivalent hydrodynamic diameter, with
the motion of smaller particles being overestimated. In reality, DLS measures the time-dependent
fluctuations in scattering intensity caused by the constructive and destructive interference resulting
from the relative Brownian movements of the NPs within a sample [33]. This is an indirect method
for the calculation of the size by means of a physical model. The calculated equivalent hydrody-
namic diameters are not compared with the particle sizes measured by direct methods, such as
SEM, TEM, and AFM. The DLS measurement requires information of the dispersant's refractive
index in which the NMs are dispersed. This is a big problem for cell culture media because there
is little information on the refractive index of these media. Operated in accordance with ISO 13321
and ISO 22412, DLS cannot distinguish between different types of particles in a suspension, and
becomes significantly less reliable for providing a wide particle size distribution because of the sixth
power dependence of the light scattering intensity on the sizes of the scattering particles. Instead,
the method produces a single average value and a number (the polydispersity index) indicating the
polydispersity of the particle population [19]. Thus, DLS is more suitable only for the measure-
ment of monodisperse particles in low-viscosity suspension, but not for polydisperse particles. To
get a better size distribution characterization of polydisperse particles in a suspension, NP track-
ing analysis (NTA) (size distribution between 20 and 1000 nm, relying on the refractive index of
particles and the viscosity of solvent) [32] or disk centrifugation (size distribution between about
5 nm and about 75 μm) [34] is preferable. NTA is a method for visualizing and analyzing particles
in liquids that relates the rate of Brownian motion to particle size. The rate of movement is related
only to the viscosity of the liquid, the temperature, and size of the particle, and is not influenced
by the particle density or refractive index. The NTA system visualizes particles as point scatters
moving under Brownian motion, but this is not a resolved image and, thus, no morphology infor-
mation can be provided, as this scatter is isotropic. NTA counts particles by simultaneously video
tracking many individual particles and relating the Brownian movement to a particle size according
to the formula derived from the Stokes-Einstein equation [35]. However, there are problems with
this particle-tracking approach. To obtain an accurate individual radius measurement, an accurate
measurement of a particle's mean-squared movement must be made. As each measured movement
is a random variable, this necessitates measuring a large number. Thus, ideally, a particle needs
to be tracked over many frames [36]. NTA can be time consuming and requires some operational
skills for the adjustment of all software settings. Recently, Walker [36] reported an alternative data-
processing method to recover a particle size distribution from NP tracking data. NTA has an advan-
tage over DLS, particularly for polydisperse samples, in that it does not, in principle, suffer from an
intensity weighting effect, as large and small particles will usually be imaged to different regions
of the camera detector array. Disk centrifugation, also called differential centrifugal sedimentation
(DCS), applies rotation speeds of up to 45,000 g . The centrifuge, in the form of a disk and filled with
a density gradient fluid, is orientated in a vertical direction for analytical purposes [38]. The size of
the particles is calculated following Stokes' law, which requires knowledge of the particle's proper-
ties such as the density of particles [39]. External or internal calibration standards are required to
determine particle size [40]. DSL, NTA, and DCS are all indirect and ensemble methods; among
the analytical techniques [37,40], electron microscopy is presently the most accurate technique to
characterize the geometric properties of NMs.
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