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