Particle Characterization (Submicron, Subvisible, and Visible Particles)
Several methods are used to characterize submicron, subvisible, and visible particles (Figure 1). Our center is equipped with the following instruments:
- Wyatt Dynapro DLS system
- Brookhaven DLS system
- Wyatt Dawn Heleos II MALS system NanoSight Particle Tracking System (LM10-HS)
- Brightwell MFI instrument
- Coulter Multisizer 3 (Beckman Coulter Inc.)
- HACH Turbidimeter
- Bruker Vertex FTIR spectrometer and HYPERION FTIR Microscope
- SEM and TEM in KU EM facility
Dynamic Light scattering (DLS)
The fluctuations of light intensity scattered from particles undergoing Brownian motion is the basis for DLS. To calculate the hydrodynamic diameter, an autocorrelation function yields a diffusion constant based on the Brownian motion of the particles. Our lab is equipped with a Wyatt Dynapro DLS system that performs DLS in sample volumes as small as 30 µl in 96- or 384-well plates as well as two Brookhaven DLS systems that can also measure the Zeta potential.
Static Light Scattering
Static light scattering (SLS) measures changes in the average intensity of scattered light, and if performed at different angles and concentrations, can determine changes in the radius of gyration of a protein molecule. We have available an SE-HPLC instrument in conjunction with UV absorbance, refractive index, and multi-angle light scattering (MALS) detectors to obtain absolute molecular weights of proteins and other macromolecules as well as soluble protein aggregates. In addition, we routinely use UV optical density spectroscopy to monitor the presence of aggregation through increases in optical density at 320-350 nm. It is also possible to obtain light scattering data during fluorescence experiments by monitoring the scattered light seen at the excitation wavelength using a second photomultiplier located at 180o to the fluorescence detector. Thus, information can be obtained about association/dissociation phenomena simultaneously with fluorescence emission data.
NanoSight (LM10-HS) visualizes, measures, and characterizes most nanoparticles (50 nm-2 µm) using a finely focused laser passed through a prism edged optical flat. Due to refraction, nanoparticles in liquid are observed using a microscope under 20X magnification. Particle size, concentration, zeta potential, and aggregation can all be analyzed using this method. NanoSight measures concentration and validates all data by recording short movies of particles moving under Brownian motion. The results are observed in real time by a CCD camera, while each particle is tracked by dedicated computer software. No information on refractive index or density is needed for particles.
Micro-flow digital imaging (MFI)
A liquid sample is pumped through a flow cell and sub-visible particles (2 µm - 70 µm) are imaged and counted by a digital camera. The images are analyzed in real-time with respect to variations in the transmitted light intensity that results from the particles in solution.
A Coulter Multisizer 3 (Beckman Coulter Inc.) with an overall sizing range of 0.4-1200 µm is used to count particles and analyze their size and distribution.
A HACH Turbidimeter is used to determine turbidity, which is related to the concentration of particles in the sample.
To determine size, morphology and composition of particles, we are equipped with a Bruker Vertex FTIR spectrometer and HYPERION FTIR Microscope.
We have access to the KU Microscopy Center, which has both scanning and transmission electron microscopes with energy-dispersive X-ray analysis (EDX) capabilities.