Imaging of Fluorophores in Chromatographic Beads, Reconstruction of Radial Density Distributions and Characterisation of Protein Uptaking Processes

Abstract

A new adjustment calculus is presented to determine the true intraparticle distribution of bound protein within chromatographic beads from confocal fluorescence slice series. The calculus does not require knowledge about optical properties of different chromatographic materials like refractive index and turbidity, but it depends on a parameter which can be adjusted interactively. The algorithm is of complexity O(n) where n is the pixel number. From the reconstructed data we compute the parameters of the protein uptaking process using a model-based approach. It is demonstrated that the protein uptaking rates of the beads strongly dependent on the conditions of the fluid phase influencing the strength of protein surface interaction.

Confocal Raman Microscopy of Protein Adsorbed in Chromatographic Particles

“…When Raman spectroscopy is applied to study distribution of unlabeled protein in the chromatographic particles, it provides additional benefits as an excellent tool to characterize protein secondary structure (Figure ). In addition, spectral normalization based on characteristic Raman bands of the particle (Figures and ) provides a nondestructive experimental avenue to correct the light attenuation effect caused by RI mismatching in the particle, unlike cross-sectioning of the particle and mathematical modeling ,, used during CLSM studies. With this new approach, quantitative comparison of protein in the chromatographic particles can be possible for the two ionic strength conditions investigated in this report.…”