Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Saturday Speaker Abstracts

Automated Imaging System for Single-Molecule Analysis in Living Cells Masato Yasui 1 , Michio Hiroshima 1 , Jun Kozuka 1 , Yasushi Sako 2 , Masahiro Ueda 1,3 . 1 QBiC, RIKEN, Suita, Osaka, Japan, 3 Osaka University, Suita, Osaka, Japan. 2 Cellular Informatics Laboratory, RIKEN, Wako, Saitama, Japan, Single-molecule imaging analysis has been applied to living cells and revealed molecular mechanisms of various intracellular events. However, technical expertise has been required for both microscope operation and data analysis, which has prevented the analysis from being a standard in medical and biological research. Here, we report a newly developed apparatus for single-molecule imaging analysis in living cells, by which single molecules on the plasma membrane can be observed without manual handling. Cell searching, focusing, and image acquisition were fully automated by utilizing a machine learning method to accomplish high accuracy, efficiency, and reproducibility. Furthermore, immersion-oil feeding, drug dispensing, and setting of the multi-well sample plate were also automated to observe many cells with different experimental conditions. The apparatus demonstrated that single-molecule imaging of EGF receptors in living CHO cells were completed for a 96-well plate within one day, in which about 600 cells were observed and analyzed automatically. Results revealed that EGF receptors adopt multiple states in their diffusion on membrane and undergo the state transition upon EGF stimulations, consistent with previous reports. The working efficiency was dramatically improved, showing that the automatically comprehensive single-molecule analysis in living cells is feasible.

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