Visualize and Measure Nanoparticle Size and Concentration

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1 NTA : Nanoparticle Tracking Analysis Visualize and Measure Nanoparticle Size and Concentration 30 Apr 2015

2 NanoSight product range LM 10 series NS300 series NS500 series Dec

3 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

4 NanoSight in Practice Load sample Insert unit Observe Nanoparticles!

5 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 Region of interest Border of sensing zone Optimal viewing region

6 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. Nanoparticle Tracking Analysis

7 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

8 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 (D t ) is derived. x, y 4 2 Dt Particle diameter (sphere equivalent hydrodynamic) d is then obtained from the Stokes Einstein equation This is an absolute method, no user calibration is required. TK B Dt 3 d K B = Boltzmann Constant = Viscosity T = Temperature

9 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 nm polystyrene in water

10 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. 120 µm 10 µm 80 µm Image shows effective scattering volume in which particles are detected and counted.

11 NTA Detection Limits Size Min. size limit is related to: Concentration Min. concentration is related to: Material type Wavelength + power of illumination Sensitivity of the camera Poor statistics (Requiring longer analysis time) 10nm 40nm Max. size limit is related to: ~10 6 particles / ml Max. concentration is related to: Limited Brownian motion Viscosity of solvent nm Inability to resolve neighboring particles Tracks too short before crossing occurs ~10 9 particles / ml 6

12 3D Plots (Size vs Intensity vs Number) 2D size v. number 2D size v. intensity 3D size v. Intensity v. concentration NanoSight has the unique ability to plot each particle s size as a function of its scattered intensity

13 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.

14 NTA Fluorescent Measurement HS camera Choice of clear or fluorescent filter: With clear filter, all light transmitted With fluorescent filter, scattered light blocked Microscope Objective Particles scatter the laser beam and if fluorescent will also fluoresce

15 Fluorescence Option 405nm 488nm 532nm 642nm Laser diodes capable of exciting fluorophores and quantum dots Optical Filters to monitor specific nanoparticles.

16 Why Fluorescence? Complex media no filter Complex media applying appropriate filter This allows for selective visualization of fluorescently- labelled particles in complex media Excitation wavelength nm Using a 565 nm Long pass optical filter

17 NTA and DLS Bimodal Sample Polystyrene reference spheres in water (100 nm and 200 nm) 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

18 NTA and DLS NTA (red profiles) and DLS (blue bars) for mixtures of polystyrene of different sizes 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: /s

19 NanoSight Biotechnology applications: Liposomes and other drug delivery vehicles Exosomes and microvesicles (extracellular vesicles) Polymers and colloids Viruses, vaccines and virus-like particles (VLPs) Gene Therapy Protein aggregates Nanoparticle toxicology Biotechnology makes up 70% of total NanoSight applications

20 NTA devices worldwide mapping 900+ instruments publications 18