Nanoparticle Tracking Analysis (particle by particle technique)
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NanoSight visualizes, measures and characterizes virtually all nanoparticles. Pls contact A&P Instrument Co.Ltd in Hong Kong for detail. Email: anson@anp.com.hk
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Nanoparticle Tracking Analysis (particle by particle technique)
- 1. www.nanosight.com Characterisation of Nanoparticles and Aggregates using Nanoparticle Tracking Analysis (NTA)
- 2. LM10 Series NS300 NS500 The Instrument Range
- 3. Mai 13 As the schematic below shows, the NanoSight technology comprises: • a metallised optical element • illuminated by laser beam . Laser as seen at low magnification NanoSight LM20 device www.nanosight.com Hardware: laser illuminated sample chamber + optical microscope
- 4. Fast and easy to use system Loading of 200nm latex standard Mai 13 www.nanosight.com
- 5. Titanium Dioxide Mai 13 www.nanosight.com Polydisperse titanium dioxide particles as seen at standard magnification
- 6. Particles are Visualised, not imaged Particles are too small to be imaged by the microscope Particles seen as point scatterers moving under Brownian motion Larger particles scatter significantly more light Speed of particles varies strongly with particle size Microvesicles purified from serum by ultracentrifugation Mai 13 www.nanosight.com
- 7. • Nanoparticles move under Brownian movement due the random movement of water molecules (red molecules in movie) surrounding them. • Small particle move faster than larger particles. • Diffusion Coefficient can be calculated by tracking the movement of each particle and then through application of the Stokes-Einstein equation particle size can be calculated. Principle of Measurement Mai 13 www.nanosight.com
- 8. • Brownian motion of each particle is followed in real-time via video • Tracking software establishes mean square displacement and diffusion coefficient (Dt) • Then from Stokes Einstein can be obtained particle (sphere equivalent hydrodynamic) diameter, dh KB = Boltzmann Constant T = Temperature = viscosity KBT Stokes-Einstein equation Dt = 3 dh Sizing is an absolute method Mai 13 www.nanosight.com Dt yx 2 4 ,
- 9. Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample. This provides a distinct advantage in determining particle size analysistrackingcapture Nanoparticle Tracking Analysis Mai 13 www.nanosight.com
- 10. The NanoSight NTA 2.0 (nano-particle tracking) analysis suite allows for captured video footage to be simultaneously tracked and analysed… 100+200nm nanoparticles being tracked and analysed by NanoSight NTA 2.0 Particle Sizing in action - Software Analysis Mai 13 www.nanosight.com
- 11. Minimum size limit is related to: • Material type • Wavelength and power of illumination source • Sensitivity of the camera Size Concentration Minimum concentration is related to: • Poor statistics (Requiring longer analysis time) 10 – 40 nm 1 000 – 2 000 nm Maximum Size limit is related to: • Limited Brownian motion • Viscosity of solvent Approx 105-6 / mL Maximum concentration is related to: • Inability to resolve neighboring particles • Tracks too short before crossing occurs Approx 109 / mL NTA Detection Limits www.nanosight.com
- 12. True size distribution profile Mixture of 100nm and 200nm latex microspheres dispersed in water in 1:1 ratio Particle distribution displays a number count vs particle size. Mai 13 www.nanosight.com
- 13. Mai 13 Number count of particle in a determined volume : Absolute concentration measurement in 10E8 part/mL. Particles are Counted– By Number Count www.nanosight.com Global concentration of all populations Concentration of a population selected by user
- 14. 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 Relative intensity scattered Mai 13 www.nanosight.com
- 16. 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. Resolving mixtures of different particle types and sizes Mai 13 www.nanosight.com
- 17. NanoSight systems can be fitted with • Choice of long pass or band pass filters allow specific nanoparticles to be tracked in high backgrounds Applications in: • Nanoparticle toxicity studies • Nano-rheology • Bio-diagnostics • Phenotyping specific exosomes • Laser diode capable of exciting fluorophores and quantum dots 405nm 488nm 532nm 635nm Fluorescent Mode available Mai 13 www.nanosight.com
- 18. Analysis of 100 nm Fluorescence standard particles suspended in FBS Mai 13 www.nanosight.com Modal particle size peaks: 103 nm Concentration: 9.5 x108 particles/ml • 100 nm fluorescently labelled particles suspended in 100% FBS • Sample maintained at a constant temperature (37 oC) • Sample viscosity – 1.33 cP • Excitation wavelength - 532 nm • Using a 565 nm Long pass optical filter No filter 565 nm Long pass optical filter This allows selective visualising of fluorescent/fluorescently labelled particles
- 19. Dynamic Light Scattering (DLS or PCS) Mai 13 www.nanosight.com • DLS (alternatively known as Photon Correlation Spectroscopy (PCS)) is deservedly an industry standard technique and widely used for 40 years... • Also relies on Brownian motion and relates the random motion of particles in liquid suspension to a hydrodynamic radius. Technique relates the rate of change of the intensity of the speckle to particle size using the auto-correlation function. • Very effective technique for measuring particle size for mono- dispersed samples from a few nm up to microns . • Widely used, big installed base.
- 20. Comparison of different particle size distribution Particle by particle method Static light scattering Dynamic Light Scattering
- 21. DLS Analysis and NanoSight equivalent for monodisperse systems NTA vs DLS - Monodisperse Mai 13 www.nanosight.com DLS NTA
- 22. With NanoSight, the analysis shows both 100nm and 200nm peaks Polystyrene reference spheres in water (100nm and 200nm) NTA vs DLS – Bimodal Sample Mai 13 www.nanosight.com
- 23. 100nm particles not seen due to intensity bias Mixed Sample of 100nm+200nm particles at a ratio of 10:1 by weight ? Mai 13 NTA vs DLS – Bimodal Sample www.nanosight.com
- 24. 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) Mixtures of polystyrene of different sizes DLS (blue bars) Mai 13 www.nanosight.com NTA vs DLS in publications
- 25. • NTA is not intensity-weighted towards larger particles, unlike DLS • While DLS is an ensemble technique, NTA operates particle-by-particle • NTA has much higher resolving power with respect to multimodal and polydisperse samples and heterogenous/mixed sample types • NanoSight unique and direct view visually validates the particle size distribution data • Number vs. Intensity vs. Size is provided for each particle size class • NTA provides particle concentration information • Fluorescence mode • Zeta Potential, particle by particle • NTA requires no information about collection angle, wavelength or solvent refractive index , unlike DLS NanoSight’s Advantages Vs. DLS/PCS Mai 13 www.nanosight.com
- 26. Comparing different Technologies Mai 13 www.nanosight.com Technique Strengths Weaknesses Electronic microscope (TEM, SEM) -Higher magnification, greater resolution, compositional information, topography and morphology -High costs - Complex sample preparation procedure Analytical Ultra Centrifugation -High resolution -High cost , complex sample preparation & data evaluation , -Low sample throughput Coulter Counters (i.e IZON) -Very low cost. - Small size -Need calibration -Surface charge of particle influences result -Clogging of aperture is common Flow Cytometers -High Throughput -Sorting of different size of particle -Fluoresence -Not suitable for vesicles sizing due to no accurate refractive index to calibrate -Lower limit is about 300nm Dynamic Light Scattering -suited to mono-disperse -samples from a few nm to a few microns -Intensity-biased Plot -No Fluorescence and no counting ability
- 27. Mai 13 Application Example : Exosomes www.nanosight.com •Microvesicles - typically 100nm to 1um range •Nanovesicles or Exosomes - 30-100nm •Originally thought to be cellular debris •Now appreciated that these cell-derived particles are of significant importance and play an important role both in cellular communication and signalling •Recently found to be present at significantly elevated levels in a number of diseases conditions Exosomes and Nanovesicles
- 28. Mai 13 Application Example : Exosomes www.nanosight.com Why use NanoSight in this application ? • We COUNT particles – concentration is key to this application. Researchers can not get good concentration information any other way – this is KEY. Remember an increase in the number of exosomes could be related to the onset of disease. •We can work in FLUORESCENCE. This means they can fluorescently label these particles and therefore SPECIATE – i.e. They know which exosomes they are looking at. •High resolution SIZE. •All of these parameters in a SINGLE MEASUREMENT – allows them to determine the size, concentration and type of exosome population.
- 29. Mai 13 Application Example : Exosomes www.nanosight.com Comparisons of different techniques 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
- 30. Mai 13 Application Example : Exosomes (Fluoresence) www.nanosight.com
- 31. Mai 13 Application Example : Purified Influenza Virus www.nanosight.com Why Nanosight? •Manufacturer interested in the purity of their product – i.e. The size distribution of particles within a sample. •Manufacturer interested in the dosage of the vaccine i.e. How many viruses are present? •Manufacturers need to know the ratio of infective to non-infective viruses in their sample. •Manufacturer interested in detecting specific viruses as the samples are purified – therefore fluorescence is of interest to them. •COUNTING aspect is the most important feature as manufacturers often use long winded infectivity assays to characterise their samples.
- 32. Current methologies for counting such as plaque assay only count infectious particles which often represent a small component in attenuated vaccines i.e. perhaps only 1% of product remains infective. The ability to count viruses in liquid suspension is essential for those working in vaccine development. Particle aggregation and yield quality are factors which need to be understood when developing these viral vaccines. Mai 13 Application Example : Purified Influenza Virus www.nanosight.com
- 33. • NanoSight technology has a unique application in the detection of early stage aggregation in protein therapeutics • Protein monomer is too small to be individually resolved by this technique, but early stage aggregates are readily detected • Protein monomer at high concentration causes high background noise in image, with the aggregate forming the resolvable particles • Both size and number of aggregates can be calculated and studied, providing insight into product stability. Data reproduced from Filipe, Hawe and Jiskoot (2010) Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2 Mai 13 Application Example: Protein Aggregation at controlled temperature (15-55o C) www.nanosight.com
- 34. • DLS analysis of aggregated protein sample generally produces a bimodal analysis. • The protein monomer and the very large aggregates get picked up through DLS as these are the regions which scatter most light. The monomer scatters a lot of light by virtue of its high concentration and the larger particles by virtue of their size. • Clearly a protein sample cannot aggregate from monomer to large micron sized aggregates with nothing in between. Whilst NanoSight cannot measure the monomer it can provide valuable information in the 30nm and above range which is typically the region which is poorly served by alternative techniques. Mai 13 Application Example: Protein Aggregation: NTA vs DLS www.nanosight.com
- 35. Mai 13 Application Example: Drug Delivery system www.nanosight.com The size will affect its 1.Circulation and residence time in the blood, 2. the efficacy of the targeting, 3. the rate of cell absorption Advantage of using Iiposome as a drug deliver • Drugs can be targeted to specific areas by attaching ligands to the liposome • Readily absorbed by cells • The rate of drug release can be controlled • Allows potentially lower doses of drug to be used, reducing toxicity and side-effects.
- 36. • Multi-walled Carbon nanotubes • Cosmetics • Foodstuffs • Ink jet inks and pigment particles • Ceramics • Fuel additives • Liposomes and other drug delivery vehicles • Virus and VLP samples • Exosomes and nanovesicles • Nanobubbles • Magnetic Nanoparticles • Precursor chemical for wafer fabrication. • Quantum dots • Polymers and colloids • CMP Slurries Mai 13 The NanoSight system is widely applicable www.nanosight.com
- 37. Size Number or concentration Polydispersity – true PSD “Relative Light Intensity” Fluorescence Surface Potential, Zeta Potential + + + + Parameters measured – simultaneously, ‘real time’, particle-by-particle... Mai 13 www.nanosight.com
- 38. • BASF, Europe • BP Castrol, Europe • DuPont, USA • Epson, Japan • Exxon Mobile, USA • GlaxoSmithKline, Europe • Merck, USA • Medimmune • Nestle, Europe • NIST, USA Selected Users • Novartis, Europe • Proctor & Gamble, USA • Roche, Europe • Smith & Nephew, Europe • Solvay, Europe • Toshiba, Japan • Unilever, Europe • US EPA • US Forces • Wyeth Biopharma, USA
- 39. NTA devices worldwilde mapping Mai 13 www.nanosight.com > 500 devices ≈ 800 publications
Editor's Notes
- 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.
- 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 bubblesAS the sample fills the cell the red laser beam emerges into from the right to the left hand side of the chamberAt 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 secondsThe 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.
- 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 scatterersIn between those sizes we see a range of diffraction patterns (Mie scatter, Airey rings)That nothing is bigger than 1micron
- Please note the viruses 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 scatteres 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.
- 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.
- 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.
- Unlike standard industry particle analysis NanoSight tracks the movement of each individual nanoparticle/virus undergoing Brownian motion to establish particle size, this is known as NanoParticle Tracking Analysis (NTA) and is unique and patented by NanoSight.Our specially designed 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 particleAnalysis: Application of the Stokes Einstein equation means that particle size can be calculated for each particle.
- 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 for1. automatic optimisation of camera settings,2. automatic rejection of particle tracks which cross,3. automatic adjustment of optimum track lengthselection,4. image noise reduction,5. sample drift correction• All these features contribute to a reproducible andaccurate nanoparticle measurement system.What information does the NTA AnalyticalSoftware 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, andoutput to spreadsheet Full reporting and batch processing facilities
- 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.
- Unlike any other method NanoSight are able to offer an approximate number count for sub micron particle and is more accurate that 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 x x20 microns so we know we are counting within a discrete area. The typical concentration range is 10^6 – 10^9particles/ml above this you 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 DLS/PCS as they graph based on light intensity.
- Unlike any other method NanoSight are able to offer an approximate number count for sub micron particle and is more accurate that 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 x x20 microns so we know we are counting within a discrete area. The typical concentration range is 10^6 – 10^9particles/ml above this you 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 DLS/PCS as they graph based on light intensity.
- 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.
- Here is the scatter intensity data added as a third axis, giving improved resolution. This is a unique capability of NanoSight.
- 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.
- This slide compares the two systems results for a monodisperse sample . As you can see the NanoSight technique has good correlation with DLS for mono-disperse samples.The only difference is NanoSight can produce an estimated of number count
- And so to a biomodal dispersion :Here we have a mixed sample of 100 and 200 nm particles. The video shows a the 2 particle types in the sample with the 200 nm particles being brighter and slower moving. You can definitely see with NanoSight that there are two sub populations of particles
- With the bimodal sample the PCS/DLS misses the 100nm particles because it assesses scatter intensity and the signal from the 100’s is much less than the 200nm particles. The larger particles therefore occlude the smaller ones: you can equate this as looking for a candle light in amongst a flood light
- With the bimodal sample the PCS/DLS misses the 100nm particles because it assesses scatter intensity and the signal from the 100’s is much less than the 200nm particles. The larger particles therefore occlude the smaller ones: you can equate this as to looking for a candle light within a flood light beam
- Direct view of what is in sample – this is unique to NanoSight. Seeing is believing.Not an average, but a particle-by-particle count – we are tracking each individual particle and are able to give you number, intensity and sizeFar less intensity weighted to larger particles – making the instrument perfect for polydisperse samples but also great to check your DLS results particularly if you are using the instruments for QA purposes whether mono or polydisperse' – note we went into one lab and found their apparently pure lab water was contaminated with so many particles three years worth of results were incorrect!The NanoSight NTA 2.0 software allows user to watch the particle size distributions grow as the analysis progresses through the captured footageThere is no requirement for inputting information on angle, wavelength or solvent refractive index.DLS is great for QA as samples there tend to be very stable but for troubleshooting in process development, R&D labs benefit from NanoSight.Summary of the advantages ofNTA over DLS NTA conducts particle-by-particle analysis, compared to DLS’saverage. So NTA avoids DLS’s bias towards larger particles NTA is much better for poly-disperse systems, where NTA canbetter resolve several peaks in a sample population. NTA can detect at much lower concentrations, down to 107particles per ml. NTA gives a good estimate of concentration, DLS cannot. NTA does not need calibration, and knowledge of refractiveindex is not required (which is a key weakness in DLS). NanoSight images are rich in information, and they provide aquick and easy way to check a sample and validate the resultsAdvantages of DLS over NTA DLS samples many more particles. DLS is ideally suited to monodispersed particlesystems. DLS has a wider size range (3-6,000nm comparedto NTA's 10-1,000nm).
- NanoSight is widely applicable. Contact us if your particles not appear on this list.