title: High throughput inclusion body sizing: Nano particle tracking analysis.
abstract: The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle-based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non-fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non-fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle-based, fast and high throughput method (1000-3000 particles), thus constituting a much more accurate and representative analysis than currently available methods.
authors.affiliation: Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Wieland N
authors.initials: WN
authors.lastname: Reichelt
authors.affiliation: Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kaineder
authors.affiliation: Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Brillmann
authors.affiliation: Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Neutsch
authors.affiliation: Division of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Taschauer
authors.affiliation: Research group electronic media, Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
authors.forename: Hans
authors.initials: H
authors.lastname: Lohninger
authors.affiliation: Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.|Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
mesh_terms: D003600:Cytosol / Q000648:ultrastructure; D002479:Inclusion Bodies / Q000737:chemistry* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D010316:Particle Size; D066329:Protein Aggregates; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D066329:Protein Aggregates
keywords: Electron microscopy; Grey scale image segmentation; Inclusion body; Nano particle tracking analysis; Stickiness