Short-cut method for the correction of light attenuation influences in the experimental data obtained from confocal laser scanning microscopy

Susanto, A.; Herrmann, Tim; Hubbuch, Jürgen

doi:10.1016/j.chroma.2006.09.053

Cited by 11 publications

(12 citation statements)

References 13 publications

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“…Since excitation and fluorescence light are attenuated in dependency of the 3D coordinate within the adsorbent particle, fluorescent intensity data will have to be corrected for light attenuation effects prior to any analysis concerning the true distribution of protein-dye conjugates within the adsorbent particle [24]. This holds true for both scanning modes commonly applied, namely horizontal and vertical scanning ( Table 3, H/V).…”

Section: Light Attenuation Effectsmentioning

confidence: 99%

“…Dealing with a variety of backbone materials and adsorbent structures, a major difficulty in the application of CLSM to chromatographic resins results from light attenuation effects caused by the material and structure of the respective adsorbent [7,9,24,25,28]. Since excitation and fluorescence light are attenuated in dependency of the 3D coordinate within the adsorbent particle, fluorescent intensity data will have to be corrected for light attenuation effects prior to any analysis concerning the true distribution of protein-dye conjugates within the adsorbent particle [24].…”

Section: Light Attenuation Effectsmentioning

confidence: 99%

“…Mathematical approaches to solve the attenuation problem apply physical models of the excitation and fluorescence emission phenomena [23,24,28,31,68,69]. In this approach, unknown model parameters (i.e., image correction parameters) are determined experimentally for the respective experimental systems without a change in system parameters.…”

Section: Light Attenuation Effectsmentioning

confidence: 99%

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Confocal laser scanning microscopy as an analytical tool in chromatographic research

Hubbuch

Kula

2008

Bioprocess Biosyst Eng

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In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.

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Section: Light Attenuation Effectsmentioning

confidence: 99%

Section: Light Attenuation Effectsmentioning

confidence: 99%

Section: Light Attenuation Effectsmentioning

confidence: 99%

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Confocal laser scanning microscopy as an analytical tool in chromatographic research

Hubbuch

Kula

2008

Bioprocess Biosyst Eng

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“…Confocal laser scanning microscopy has been proven very useful for imaging inhomogeneous densities of fluorophores, see Susanto et al (2006a) and references therein. However, imaging of spatial density distributions involves absorption effects, since radiation traveling in matter is more or less absorbed, i.e., converted into different energy forms, especially heat.…”

Section: Reconstruction Of the Spatial Densitymentioning

confidence: 99%

“…For a sound description of a robust incremental compensation of the light attenuation in 3D fluorescence microscopy applied to cellular structures is given by Kervrann et al (2004). In Susanto et al (2006a) the light attenuation effects in CLSM data of spherical beads are corrected in two steps. First, one assumes plane wave fronts perpendicular to the optical axis, and starting from the Lambert-Beer law, an updating formula for varying protein density is used basing on numerical integration over the just computed values of this density.…”

Section: Introductionmentioning

confidence: 99%

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

Stanislawski

Schmit²,

Ohser

2010

Image Anal Stereol

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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.

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Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies

Salm

Iskra

et al. 2013

Biotech & Bioengineering

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Fouling of chromatographic resins over their operational lifetimes can be a significant problem for commercial bioseparations. In this article, scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small-scale column studies were applied to characterize a case study where fouling had been observed during process development. The fouling was found to occur on an anion exchange (AEX) polishing step following a protein A affinity capture step in a process for the purification of a monoclonal antibody. Fouled resin samples analyzed by SEM and batch uptake experiments indicated that after successive batch cycles, significant blockage of the pores at the resin surface occurred, thereby decreasing the protein uptake rate. Further studies were performed using CLSM to allow temporal and spatial measurements of protein adsorption within the resin, for clean, partially fouled and extensively fouled resin samples. These samples were packed within a miniaturized flowcell and challenged with fluorescently labeled albumin that enabled in situ measurements. The results indicated that the foulant has a significant impact on the kinetics of adsorption, severely decreasing the protein uptake rate, but only results in a minimal decrease in saturation capacity. The impact of the foulant on the kinetics of adsorption was further investigated by loading BSA onto fouled resin over an extended range of flow rates. By decreasing the flow rate during BSA loading, the capacity of the resin was recovered. These data support the hypothesis that the foulant is located on the particle surface, only penetrating the particle to a limited degree. The increased understanding into the nature of the fouling can help in the continued process development of this industrial example.Scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small-scale column experiments were applied to characterize a case study where fouling had been observed on an anion exchange chromatography in a monoclonal antibody process. The results suggest the foulant is located on the particle surface, resulting in a minimal decrease in saturation capacity, but having a significant impact on the kinetics of adsorption, severely decreasing protein uptake rate.

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Short-cut method for the correction of light attenuation influences in the experimental data obtained from confocal laser scanning microscopy

Cited by 11 publications

References 13 publications

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Confocal laser scanning microscopy as an analytical tool in chromatographic research

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

Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies

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