title: Variable Threshold Method for Determining the Boundaries of Imaged Subvisible Particles.
abstract: An accurate assessment of particle characteristics and concentrations in pharmaceutical products by flow imaging requires accurate particle sizing and morphological analysis. Analysis of images begins with the definition of particle boundaries. Commonly a single threshold defines the level for a pixel in the image to be included in the detection of particles, but depending on the threshold level, this results in either missing translucent particles or oversizing of less transparent particles due to the halos and gradients in intensity near the particle boundaries. We have developed an imaging analysis algorithm that sets the threshold for a particle based on the maximum gray value of the particle. We show that this results in tighter boundaries for particles with high contrast, while conserving the number of highly translucent particles detected. The method is implemented as a plugin for FIJI, an open-source image analysis software. The method is tested for calibration beads in water and glycerol/water solutions, a suspension of microfabricated rods, and stir-stressed aggregates made from IgG. The result is that appropriate thresholds are automatically set for solutions with a range of particle properties, and that improved boundaries will allow for more accurate sizing results and potentially improved particle classification studies.
authors.affiliation: Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, Maryland 20899. Electronic address: rcavicchi@nist.gov.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Cavicchi
authors.affiliation: West Virginia Wesleyan College, Buckhannon, West Virginia 26201.
authors.forename: Cayla
authors.initials: C
authors.lastname: Collett
authors.affiliation: Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
authors.forename: Srivalli
authors.initials: S
authors.lastname: Telikepalli
authors.affiliation: Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Zhishang
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
authors.forename: Michael
authors.initials: M
authors.lastname: Carrier
authors.affiliation: Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
authors.forename: Dean C
authors.initials: DC
authors.lastname: Ripple
mesh_terms: D000465:Algorithms; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry*; D010316:Particle Size; D066329:Protein Aggregates; D014867:Water / Q000737:chemistry
chemical_list: D007074:Immunoglobulin G; D066329:Protein Aggregates; D014867:Water; D005990:Glycerol
keywords: flow imaging; image analysis; microscopy; particle sizing; physical characterization; protein aggregates