Scanning Microfluorescence Analysis of Immobilized-Cell Biocatalysts

本提案的目的是开发和优化一种新的实验方法，基于扫描微荧光，将细胞生理学表征为固定细胞生物催化剂内时间和位置的函数。该方法将用于研究固定化大肠杆菌细胞内质粒的稳定性。该方法采用了一种生物催化剂结构，可以直观地观察被困在离营养源不同距离的细胞，从而可以观察到营养良好的细胞和饥饿的细胞。还使用了最近开发的扫描微荧光仪。该仪器具有先进的图像分析能力，其激发激光束可以聚焦到直径小于1微米。这些特征允许测量单个细菌细胞的荧光。质粒的稳定性将使用附着在目标质粒上的荧光标记基因来监测。这种新的分析方法既强大又通用。通过改变荧光染色，可以研究多种固定细胞现象。研究结果将增强对固定化细胞生理学的基本理解，并允许对工业上重要的固定化细胞过程的数学模型进行改进。

The object of this proposal is to develop and optimize a novel experimental approach, based on scanning microfluorescence, to characterize cellular physiology as a function of both time and position within immobilized-cell biocatalysts. The approach will then be used to investigate plasmid stability within immobilized Escherichia coli cells. The method employs a biocatalyst configuration which allows visual observation of cells entrapped at various distances from the nutrient supply, so that well-nourished, as well as starving cells may be viewed. A recently developed scanning microfluorescence instrument is also used. This instrument has advanced image- analysis capabilities, and its excitation laser beam can be focused to a diameter less than one micron. These features allow fluorescence of individual bacterial cells to be measured. Plasmid stability will be monitored using fluorescent marker genes attached to target plasmids. This new analytical approach is both powerful and versatile. A wide variety of immobilized-cell phenomena can be studied by changing fluorescent stains. Results of the study will enhance the fundamental understanding of immobilized-cell physiology and allow refinements in mathematical models for industrially important immobilized-cell processes.