Confocal laser scanning microscopy as an analytical tool in chromatographic research.
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.
Hubbuch
Jürgen
J
Institut für Biotechnologie 2, Forschungszentrum Jülich, 52425 Jülich, Germany. j.hubbuch@fz-juelich.de
Kula
Maria Regina
MR
D000327:Adsorption; D000465:Algorithms; D001709:Biotechnology / Q000379:methods; D002845:Chromatography / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods*; D008968:Molecular Conformation; D038761:Photobleaching; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011506:Proteins