30 Apr 2015
NTA : Nanoparticle Tracking Analysis
Visualize and Measure Nanoparticle Size and
Concentration
NanoSight product range
LM 10 series NS300 series NS500 series
Dec 13 www.nanosight.com 34
NanoSight Technology As the schematic below shows, the NanoSight
technology comprises:
• a proprietary optical element
• illuminated by a laser beam
Above: the laser as seen at low
magnification
Below: the NanoSight Viewing Cell
.
NanoSight in Practice
Load sample Insert unit Observe Nanoparticles!
NanoSight System in Action
Region at which illumination
beam emerges into sample
Border of sensing zone
Optimal viewing region
View of beam passing through sample and zooming into desired field of view.
Region of
interest
Nanoparticle Tracking Analysis Nanoparticle Tracking Analysis (NTA) measures particle size by video analysis
of the Brownian motion, simultaneously, of many individual particles.
This results in a particle size distribution of high resolution, particle concentration
and an ability to include additional particle characteristics such as relative light
scattering intensity or fluorescence.
Analysis Tracking Nanoparticle
Principle of Measurement • Nanoparticles move under
Brownian motion
• Small particles move faster than
larger particles
• Diffusion Coefficient can be
calculated by tracking and
analysing the movement of each
particle separately but
simultaneously
• Through application of the
Stokes-Einstein equation,
particle size can be calculated
• The scattering or fluorescence
properties of particles is also
measured
• Particle concentration can also
be obtained
NTA Sizing: an Absolute Method
› Brownian motion of each particle is
followed in the real-time video
› Particle tracking software measures
mean square displacement in two
dimensions and diffusion coefficient
(Dt) is derived.
› Particle diameter (sphere equivalent
hydrodynamic) d is then obtained from
the Stokes Einstein equation
› This is an absolute method, no user
calibration is required.
KB = Boltzmann Constant
= Viscosity
T = Temperature
Dt
yx
2
4
,
d
TKDt B
3
NanoSight Provides a Visualisation of the
Light Scattered by Nanoparticles
not a Resolved Image of the Particles
• Particles are too small to be
imaged by microscope
• Small particles are visualised as
point scatterers moving under
Brownian motion
• Larger particles scatter significantly
more light
100+200nm polystyrene in water
80 µm
10 µm
120 µm
Image shows effective scattering
volume in which particles are
detected and counted.
Concentration
• Small sample volume from 0.3 ml required.
• The concentration estimate is measured per
unit time (i.e. an average over the course of
the video)
• Therefore, particles moving in and out of the
field of view do not affect concentration
estimate.
6
Min. size limit is related to:
› Material type
› Wavelength + power of illumination
› Sensitivity of the camera
Size Concentration
Min. concentration is related to:
› Poor statistics (Requiring longer analysis
time)
10nm – 40nm
1000-2000nm
Max. size limit is related to:
› Limited Brownian motion
› Viscosity of solvent
~106 particles / mL
Max. concentration is related to:
› Inability to resolve neighboring particles
› Tracks too short before crossing occurs
~109 particles / mL
NTA Detection Limits
3D Plots (Size vs Intensity vs Number)
NanoSight has the unique ability to plot each particle’s size as a function
of its scattered intensity
2D size v.
number 2D size v.
intensity
3D size v.
Intensity v.
concentration
Resolving different materials mixtures
100 PS
60nm Au
30nm Au
In this mixture of 30 nm and 60 nm gold nanoparticles mixed with 100 nm
polystyrene, the three particle types can be clearly seen in the 3D plot confirming
indications of a tri-modal given in the normal particle size distribution plot. Despite
their smaller size, the 60 nm Au can be seen to scatter more than the 100 nm PS.
NTA Fluorescent Measurement
HS
camera
Microscope
Objective Particles scatter the laser beam and if fluorescent will also fluoresce
With clear filter, all light transmitted
With fluorescent filter, scattered light blocked
Choice of clear or fluorescent filter:
405nm 488nm 532nm 642nm
• Laser diodes capable of exciting fluorophores and quantum dots
• Optical Filters to monitor specific nanoparticles.
Fluorescence Option
• Excitation wavelength - 532 nm
• Using a 565 nm Long pass optical filter
Complex media – no filter Complex media – applying appropriate filter
This allows for selective visualization of
fluorescently- labelled particles in complex media
Why Fluorescence?
Polystyrene reference spheres
in water (100 nm and 200 nm)
NTA and DLS – Bimodal Sample
In NTA, analysis clearly shows
both 100nm and 200nm peaks
NTA can get down to 1:1.25 ratio DLS has a resolution ratio of 1:3
Data reproduced from Filipe, Hawe and Jiskoot (2010) “Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates”, Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2
NTA (red profiles)
and DLS (blue
bars) for mixtures
of polystyrene of
different sizes
NTA and DLS
› Viruses, vaccines
and virus-like
particles (VLPs)
› Gene Therapy
› Protein aggregates
› Nanoparticle
toxicology
› Liposomes and
other drug delivery
vehicles
› Exosomes and
microvesicles
(extracellular
vesicles)
› Polymers and
colloids
NanoSight Biotechnology applications:
Biotechnology makes up 70% of total NanoSight applications
NTA devices worldwide mapping
18
900+ instruments
1500+ publications
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