1. Nanoparticle Tracking Analysis Multi-parameter Characterization at NanoScale 產品專員 許勝淵 / 施美琪

2. The Instrument Range NS300 LM10 Series LM20 NS500
There are two core instruments: the LM10 which a microscope based system and the LM20 which you see here which does not have microscope oculars but has the microscope optics internally. The two systems are identical in performance regarding software:
LM10 has an option to view particles at lower magnification with the additional of a high sensitivity camera for applications such as Virology
LM20 is just easier to use for non microscopers and in multi-user environments :
The viewing field is optimised and located automatically.
Temperature measurement can be integrated
The hardware consists of :
Dependent on the model a full microscope on the LM10 or the microscope objectives for the LM20
Laser encased in an optical sample chamber of 0.25ml volume
CCD camera – this generates a AVI file
NTA 2.0 Nanoparticle Tracking Analysis software
LM10 Series
LM20
NS500

3. NanoSight’s Technology
Above: the laser beam as seen at low magnification
Below: A NanoSight Viewing Cell
The above image shows a schematic of the laser sample chamber.
Within the laser sample unit is a 35mW 638nm red laser diode, a glass prism and a top plate which forms a seal with the glass optic.
The top plate and glass optic sandwich a layer of liquid between them which contains the sample.
The laser beam hits the angled surface of the glass optic at 90 degrees and as such passes straight through this leading edge. The laser beam then propagates from the glass optic into the liquid layer. It enters the liquid layer from the glass at and angle and due to a difference in RI between the glass and liquid, the laser beam will refract across the surface of the glass optic.
The high power density, high SNR laser beam passes through the samples and the nanoparticles scatter light which is collected using the microscope objective.
As the schematic above shows, the NanoSight technology comprises:
a proprietary optical element
illuminated by specially configured laser beam

4. NanoSight in Practice Load sample Insert unit Observe Nanoparticles!
This video demonstrates the instrument in use and how easy it is to load a sample:
Inject the sample using a syringe through the luer ports into the sample chamber: this must be done slowly and vertically to avoid air bubbles
As the sample fills the cell the red laser beam emerges into from the right to the left hand side of the chamber
At this point you can do an eyeball check on your sample: if the beam is “thick” then there will be lots of particles and it is likely your sample is too concentrated. You ideally want to see a thin red line.
Slot the sample chamber back onto the main instrument and there is instant live feed from the camera to the PC and you can visualise your samples immediately.
In total this takes about 10 seconds
The sample chamber is also easy to clean, and can simply be flushed through when using the same solvent. Cleanliness is clear in the view the instrument provides.
Load sample
Insert unit
Observe Nanoparticles!

5. The NanoSight System in Action
View of beam passing through sample and zooming into desired field of view.
Region at which illumination beam emerges into sample
Border of sensing zone
Optimal viewing region
Region of interest
This image shows the typical images when viewed through the eyepieces of the LM10.
The red colour is associated with the laser beam used to illuminate the particles and is not indicative of the colour of the particles.
The region which looks like a thumbprint is the point at which the laser beam emerges at the glass liquid interface, the laser beam refracts from this point from top to bottom in the field of view. The optimal region for imaging particles is the point just after the laser beam has emerged into the liquid phase. This optimal viewing position is obtained automatically with the LM20 instruments.

6. An Immediate Visualization of Particles
An idea of the presence, size, polydispersity and concentration of nanoparticles can be obtained immediately on loading of sample
This TiO2 sample shows clear polydispersity
The particles sizes are approximately 60 nm to 800 nm
Here is a polydisperse sample of Titanium Dioxide in water in NanoSight video format.
The image is information rich and the user can establish many things about their sample at a glance:
The largest particle is about 500nm we know this because we are beginning to see its shape ie its no longer truly spherical.
The smallest ones are around 60 – 70 nm and are point scatterers
In between those sizes we see a range of diffraction patterns (Mie scatter, Airey rings)
That nothing is bigger than 1micron
Titanium Dioxide (in water)

7. Particle Size and Size Distribution
Measurement Parameters
Particle Size and Size Distribution
Particle Count and Concentration
Fluorescence mode
Plus information on:
Composition/structure (materials differentiated by light scatter intensity)
Shape (an indication of degree of sphericity)
….all particle-by-particle and with unique visual validation.
Photon Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering (DLS) is an industry standard technique which relates Brownian motion to particle size.
DLS does not generate an image of the particle, rather analyses the fluctuations in light intensity caused by the movement of the particles within a sample.
Light scattered from particles is relative to the 6th power of the particle radius, hence a 200nm particle will scatter x64 more light than a 100nm particle.
As this technique looks at fluctuations in light intensity, the technique has an inherent bias towards larger particles within a sample, by virtue of the fact they scatter more light.
The technique produces an average particle size, averaged out over hundreds of thousands of particles. As such it is very well suited for analysis of mono-disperse samples. Due to the skew towards the larger particles it is less well suited for samples with larger aggregates or samples which are poly-disperse.
Accurate information with regards to particle concentration can not be achieved through DLS.

8. 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.
Unlike standard industry particle analysis (DLS/PCS) NanoSight tracks the movement of each individual nanoparticle/virus undergoing Brownian motion to establish particle size, this is NanoParticle Tracking Analysis (NTA) and is unique to NanoSight.
The NTA software determines the particle size is a 3 step process:
Capture: The software captures a movie file of the particle moving under Brownian motion.
Tracking:. The software follows the movement of each particle independently and then calculates the mean squared displacement for each particle
Analysis: Application of the Stokes Einstein equation means that particle size can be calculated for each particle.
This process can be executed “Live” without intermediate video recording.

9. Principal 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
The principle of measurement for NTA is Brownian motion. The key factors pertinent to our measurement are:
Nanoparticles move randomly under solvent bombardment at a speed related to their size:
Large particle move slowly under Brownian motion whilst smaller particles move fast under brownian motion.
Brownian motion is independent of particle density and therefore 100nm gold particles will move at the same speed as 100nm polystyrene particles.

10. NTA Sizing… is an Absolute Method
Brownian motion of each particle is followed in real-time via video
Particle tracking software measures mean square displacement in two dimensions = diffusion coefficient (Dt)
Particle diameter (sphere equivalent hydrodynamic) d is then obtained from the Stokes Einstein equation
Because this is an absolute method, no user calibration is required
The technique measures mean squared displacement from which the particle diffusion coefficient can be calculated.
The Stokes Einstein equation can then be applied to calculate particle size.
The size calculated is a sphere equivalent size.
The user needs to know the viscosity and temperature of the solvent as these parameters influence the particle diffusion.
No information is required on particle density or the solvent refractive index as the technique is independent of these parameters
NTA is an absolute method requiring no calibration.
KB = Boltzmann Constant
= viscosity
T = Temperature

11. Particle Sizing in Action
An immediate idea of sample concentration and size is gained in seconds
A live view of progress ensures optimal analysis is maintained until stable size distribution profile is obtained
This is the software in action , we are processing a recorded clip.
You can see the individual particles being tracked and a calculation being made for each track.
The longer the processing time the better the repeatability of the result – approximately 40 seconds for a mono-disperse and a twice as long for a polydisperse system.
The NTA programme
 The NTA programme allows for
1. automatic optimisation of camera settings,
2. automatic rejection of particle tracks which cross,
3. automatic adjustment of optimum track length
selection,
4. image noise reduction,
5. sample drift correction
• All these features contribute to a reproducible and
accurate nanoparticle measurement system.
What information does the NTA Analytical
Software provide?
 Real-Time dynamic nano-particle visualisation
 Particle-by-particle analysis
 Particle counting and sizing
 Particle scatter intensity vs. count and size (3D plot)
 Particle size distributions displayed as histograms, and
output to spreadsheet
 Full reporting and batch processing facilities
125 nm Liposomes

12. NTA Detection Limits - Size
Minimum Size limit is dependent upon:
Material type
Wavelength and power of illumination source
Sensitivity of the camera nm
Maximum Size limit is dependent upon:
Limited Brownian motion nm
Size range is 10 – 1000 nm (1 micron)
The lower end of the size limit is affected by the refractive index of the particle.
Also the ratio of the RI of the solvent and particle is important; if the refractive indices are too similar (“index-matched”) then no scatter occurs and particles cannot be detected or tracked.
When working with samples near the lower limit of detection, there is no subsitute for sample work, which NanoSight are always happy to undertake.

13. NanoSight provides a Visualization of the Light Scattered by Nanoparticles – this is 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
Please note the particles are not imaged directly as they are smaller than the wavelength of light and hence cannot be resolved – this is the reason you can image viruses in EM as a low wavelength electron source is used which allows much smaller particles to be resolved.
As such the technique is used to image the light scattered from the particles and not the particle itself. The particles act as point scatterers and the light scattered is used to identify the position of the particle for the purpose of tracking their movement.
No information is available on the surface characteristics of the nanoparticle.
nm polystyrene in water

14. Particle Count and Concentration
Measurement Parameters
Particle Size and Size Distribution
Particle Count and Concentration
Fluorescence mode
Plus information on:
Composition/structure (materials differentiated by light scatter intensity)
Shape (an indication of degree of sphericity)
….all particle-by-particle and with unique visual validation.
Photon Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering (DLS) is an industry standard technique which relates Brownian motion to particle size.
DLS does not generate an image of the particle, rather analyses the fluctuations in light intensity caused by the movement of the particles within a sample.
Light scattered from particles is relative to the 6th power of the particle radius, hence a 200nm particle will scatter x64 more light than a 100nm particle.
As this technique looks at fluctuations in light intensity, the technique has an inherent bias towards larger particles within a sample, by virtue of the fact they scatter more light.
The technique produces an average particle size, averaged out over hundreds of thousands of particles. As such it is very well suited for analysis of mono-disperse samples. Due to the skew towards the larger particles it is less well suited for samples with larger aggregates or samples which are poly-disperse.
Accurate information with regards to particle concentration can not be achieved through DLS.

15. Particles are Counted and Concentration is Measured
Mixture of 100 nm and 200 nm latex microspheres dispersed in water in 1:1 ratio
*(Size is not intensity weighted, as in Dynamic Light Scattering)
Unlike any other method NanoSight is able to offer an approximate number count for sub micron particles and is more accurate than a volume density calculation. This is because NanoSight tracks the individual particles and not an average as in DLS. The field screen is 120 x 80 x20 microns so we know we are counting within a discrete volume. The typical concentration range is 10^6 – 10^9particles/ml above this the user will need to dilute.
The video illustrates the accurate count of a bi-modal sample of latex spheres at 100 and 200 nm dispersed 1:1 in water. The particle count can been seen on this section of the screen The graph again is particle size versus particle concentration . This cannot be directly compared to raw DLS/PCS output which is expressed on light intensity.
Particle distribution displays a number count vs. particle size*
The particle concentration by user- selectable size class is provided

16. Particles are Counted and Concentration is Measured by Size Class
Unlike any other method NanoSight is able to offer an approximate number count for sub micron particles and is more accurate than a volume density calculation. This is because NanoSight tracks the individual particles and not an average as in DLS. The field screen is 120 x 80 x20 microns so we know we are counting within a discrete volume. The typical concentration range is 10^6 – 10^9particles/ml above this the user will need to dilute.
The video illustrates the accurate count of a bi-modal sample of latex spheres at 100 and 200 nm dispersed 1:1 in water. The particle count can been seen on this section of the screen The graph again is particle size versus particle concentration . This cannot be directly compared to raw DLS/PCS output which is expressed on light intensity.

17. NTA Detection Limits - Concentration
Minimum detectable concentration is dependent upon:
Sufficient number of particles
Insufficient number of particles = Poor statistics
approx 107/ml
Maximum concentration is related to:
Inability to resolve neighboring particles
Tracks too short before crossing occurs
approx 109/ml
Size range is 10 – 1000 nm (1 micron)
The lower end of the size limit is affected by the refractive index of the particle.
Also the ratio of the RI of the solvent and particle is important; if the refractive indices are too similar (“index-matched”) then no scatter occurs and particles cannot be detected or tracked.
When working with samples near the lower limit of detection, there is no subsitute for sample work, which NanoSight are always happy to undertake.

18. Fluorescence mode Measurement Parameters
Particle Size and Size Distribution
Particle Count and Concentration
Fluorescence mode
Plus information on:
Composition/structure (materials differentiated by light scatter intensity)
Shape (an indication of degree of sphericity)
….all particle-by-particle and with unique visual validation.
Photon Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering (DLS) is an industry standard technique which relates Brownian motion to particle size.
DLS does not generate an image of the particle, rather analyses the fluctuations in light intensity caused by the movement of the particles within a sample.
Light scattered from particles is relative to the 6th power of the particle radius, hence a 200nm particle will scatter x64 more light than a 100nm particle.
As this technique looks at fluctuations in light intensity, the technique has an inherent bias towards larger particles within a sample, by virtue of the fact they scatter more light.
The technique produces an average particle size, averaged out over hundreds of thousands of particles. As such it is very well suited for analysis of mono-disperse samples. Due to the skew towards the larger particles it is less well suited for samples with larger aggregates or samples which are poly-disperse.
Accurate information with regards to particle concentration can not be achieved through DLS.

19. NanoSight in Fluorescence Mode
Selected NanoSight Instruments are supplied with a choice of blue (404nm), green (532nm) or red (638nm) laser diodes capable of exciting fluorophores and quantum dots
Filters allow specific nanoparticles to be tracked in high backgrounds
Wide applications in:
Nanoparticle toxicity studies
Nano-rheology
Bio-diagnostics

20. Excitation/Fluorophores/Filter combination
blue
430
green
565
red
Nile red

21. Example – Detection of Fluorescent Particles in Mixture
A mixture of 50nm fluorescent particles and 200nm non-fluorescent particles analysed under light scatter mode
Same mixture when scatter removed by fluorescence filter

22. Example – Fluorescence Detection in Biological Fluids
Example of analysis of cellular micro- and nano-vesicles labelled with an appropriate fluorescent antibody.
Light scatter
Antibody Fluorescence
Particle size distribution profile of sample under light scatter (green line) and fluorescent (red line) analysis.

23. Measurement Parameters
Particle Size and Size Distribution
Particle Count and Concentration
Zeta Potential
Fluorescence mode
Plus information on:
Composition/structure (materials differentiated by light scatter intensity)
Shape (an indication of degree of sphericity)
….all particle-by-particle and with unique visual validation.
Photon Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering (DLS) is an industry standard technique which relates Brownian motion to particle size.
DLS does not generate an image of the particle, rather analyses the fluctuations in light intensity caused by the movement of the particles within a sample.
Light scattered from particles is relative to the 6th power of the particle radius, hence a 200nm particle will scatter x64 more light than a 100nm particle.
As this technique looks at fluctuations in light intensity, the technique has an inherent bias towards larger particles within a sample, by virtue of the fact they scatter more light.
The technique produces an average particle size, averaged out over hundreds of thousands of particles. As such it is very well suited for analysis of mono-disperse samples. Due to the skew towards the larger particles it is less well suited for samples with larger aggregates or samples which are poly-disperse.
Accurate information with regards to particle concentration can not be achieved through DLS.

24. 3D Plots (Size Vs. Intensity Vs. Number)
2D size Vs. number
2D size Vs. intensity
3D size Vs. Intensity Vs. concentration
Size range is 10 – 1000 nm (1 micron)
The lower end of the size limit is affected by the refractive index of the particle.
Also the ratio of the RI of the solvent and particle is important; if the refractive indices are too similar (“index-matched”) then no scatter occurs and particles cannot be detected or tracked.
When working with samples near the lower limit of detection, there is no subsitute for sample work, which NanoSight are always happy to undertake.
NanoSight has the unique ability to plot each particle’s size as a function of its scattered intensity

25. Scattered Intensity – An Addional Variable
The ability of NanoSight to plot nanoparticle size against intensity allows high resolution plots to be obtained from samples in which such information may be lost in a single plot of particle size distribution only
Size
Number
Intensity
100
200
200’s
100 dimers?
100’s
Here is the scatter intensity data added as a third axis, giving improved resolution. This is a unique capability of NanoSight. 25

26. Information About Type of Particle
50nm Gold + 100nm Latex
100nm nm Latex
Plotting size vs. relative scattering intensity (≡ particle Ri) allows differences in particle composition to be explored. Note the higher scattering but smaller size of gold vs. polystyrene (left) compared to the scaling of size to intensity for two sizes of polystyrene (right)
Here is the scatter intensity data added as a third axis, giving improved resolution. This is a unique capability of NanoSight. 26

27. Resolving Mixtures of Different Particle Types and Sizes
100 PS
60nm Au
30nm Au
Here is the scatter intensity data added as a third axis, giving improved resolution. This is a unique capability of NanoSight.
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. 27

28. Differentiating Virus in Complex Background (Growth Media)
Scatter Intensity
Growth Medium
Virus
Growth Medium
Particle Size, nm.
Growth Medium plus second population, which is virus particles. The two populations are similar sizes but are differentiated because the virus scatters more light.

29. Shape (an indication of degree of sphericity)
Measurement Parameters
Particle Size and Size Distribution
Particle Count and Concentration
Zeta Potential
Fluorescence mode
Plus information on:
Composition/structure (materials differentiated by light scatter intensity)
Shape (an indication of degree of sphericity)
….all particle-by-particle and with unique visual validation.
Photon Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering (DLS) is an industry standard technique which relates Brownian motion to particle size.
DLS does not generate an image of the particle, rather analyses the fluctuations in light intensity caused by the movement of the particles within a sample.
Light scattered from particles is relative to the 6th power of the particle radius, hence a 200nm particle will scatter x64 more light than a 100nm particle.
As this technique looks at fluctuations in light intensity, the technique has an inherent bias towards larger particles within a sample, by virtue of the fact they scatter more light.
The technique produces an average particle size, averaged out over hundreds of thousands of particles. As such it is very well suited for analysis of mono-disperse samples. Due to the skew towards the larger particles it is less well suited for samples with larger aggregates or samples which are poly-disperse.
Accurate information with regards to particle concentration can not be achieved through DLS.

30. Shape
Images of non-Spherical particles “flash” “Twinkle” (scintillate) under NanoSight:
As spherical monomer particles aggregate, the aggregates increasingly exhibit twinkling images in NanoSight
As the aspect ratio of particles exceeds around 3:1 the particles flash
Filimentous and disc-shaped particles exhibit the most pronounced flashing
Spherical particles exhibit constant scatter signals with NanoSight:
Sphericity is confirmed through Electron Microscopy
A wide collection angle eliminates any angular dependence of the scatter signal
Photon Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering (DLS) is an industry standard technique which relates Brownian motion to particle size.
DLS does not generate an image of the particle, rather analyses the fluctuations in light intensity caused by the movement of the particles within a sample.
Light scattered from particles is relative to the 6th power of the particle radius, hence a 200nm particle will scatter x64 more light than a 100nm particle.
As this technique looks at fluctuations in light intensity, the technique has an inherent bias towards larger particles within a sample, by virtue of the fact they scatter more light.
The technique produces an average particle size, averaged out over hundreds of thousands of particles. As such it is very well suited for analysis of mono-disperse samples. Due to the skew towards the larger particles it is less well suited for samples with larger aggregates or samples which are poly-disperse.
Accurate information with regards to particle concentration can not be achieved through DLS.

31. Particle Aggregation 60nm Gold
DNA mediated aggregation. Potentially provides an alternative to fluorescence based assays or signal amplification procedures such as PCR, in nucleic acid diagnostics
In the second video the aggregates are brighter and slower moving. Their increased hydrodynamic diameter is detected
Prior to aggregation
This is a DNA detection experiment is an interesting example of how our products are used and will be of particular interest to bio-nano people: This videos illustrate Gold colloids (which are commonly used in diagnostic kits) aggregated with DNA. Dna has been added to 60nm gold particles and the videos show the before and after particles
The first video shows two sets of 60nm gold particles which are tagged with different but complimentary dna fragments
When the DNA which is suppose to detects the analytes is added we see in the second image that the particles combine together into dimmers (like a dumbell shape). This increases their size and as such they are larger (81nm), slower, brighter and they now flash because they are asymmetric
The technique can follow the kinetics of aggregation in 3 ways:
Change in particle size over time.
Change in concentration of particles over time.
What the images look like over time.
81 nm
Following addition of DNA

32. Auto-Generated Report
Auto-generated Report contains:
User and sample details
Capture settings
Analysis settings
Histogram data
Graphs
Tables
Excel compatible raw data files
Here is the scatter intensity data added as a third axis, giving improved resolution. This is a unique capability of NanoSight. 32

33. Application Specific Competition:
Flow Cytometry:
Limited lower limit of detection – can’t detect the smaller exosomes
DLS:
Samples are often not very homogenous – therefore not suited for measurement with DLS.
DLS cannot count and also can’t phenotype the exosomes – meaning it has limited value.
EM:
Time consuming and expensive

34. KEY USERS: MedImmune (West US- Santa Clara)
Merial, France, Merial USA
NanoViricides Inc
NIAID/NIH Vaccine Research center
Novartis Vaccines & Diagnostics
Pall Europe
Pfizer Inc - Andover MA (Formerly Wyeth)
Purdue University
Tokyo University
Transgene
UCL University College London (1) - Chem Eng
UCL University College London (2) - Biochemical Eng
University of Cambridge - Engineering Dept
University of Florida - Pathology
University of Leicester (2) - Dept of Infection
University of Strathclyde (1) - Pharmacy/Biomedical
University of Vienna (Bodenkultur)
Walter Reed Army Institute of Research
Abbott Biologicals, Netherlands
Baxter AG
BIA Separations d.o.o.
Big DNA (Was Moredun)
Biocompatibles UK Ltd
Biogen Idec (CA)
Biogen Idec (MA)
BN ImmunoTherapeutics
Clinical Biomanufacturing Facility, Oxford University
Eden BioDesign
GE Healthcare
GenVec
GSK GlaxoSmithKline, USA
Henderson Morley, UK
IDT Biologika GmbH
Intercell
Intervet
LifeCycle Pharma A/S
MedImmune (West US- Mountain View)

35. Further reading/ 3rd party papers:
X113 Shangfeng Du, Kevin Kendall, Susan Morris, Clive Sweet (2010) Measuring number-concentrations of nanoparticles and viruses in.pdf (3rd party paper)
X170 Kevin Kendall et al, Virus concentration and adhesion measured by laser tracking.pdf (3rd party paper)
AndersonBP1-2.pdf (3rd party paper)
M109B Application Note Virus.pdf (application note)
M114D Application Note Comparison of Infectivity Assays and NTA.pdf (application note)

36. Thanks for your attention!