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09월 05일 17시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능
제120회 대한화학회 학술발표회, 총회 및 기기전시회 안내
Automated single-molecule imaging and its applications to cell signaling
2017년 9월 6일 11시 13분 42초
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목 10시 : 35분
Analytical Chemistry-Life Chemistry -
[Analytical Chemistry Division - JASIS (Japan) - Life Chemistry Division Joint Symposium] Super-Resolution Optical Microscopy and Single-Cell Analysis
Graduate School of Frontier Biosciences, Osaka University / Quantitative Biology Center (QBiC), RIKEN, Japan
Single-molecule imaging analysis has been applied to living cells and revealed molecular mechanisms of various intracellular events. The techniques have made it possible to directly monitor the behaviors of biomolecules in living cells, in which the locations, movements, turnovers, and complex formations of biomolecules can be detected quantitatively with single molecule sensitivity, providing powerful tools to elucidate molecular mechanisms of intracellular signaling processes. 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 molecules on the plasma membrane can be observed at single-molecule level 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 containing cells were also automated to observe many cells with different experimental conditions. The apparatus demonstrated that single-molecule imaging of EGF receptors in the living CHO cells were completed for a 96-well plate within one day, in which about 1000 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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