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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Saturday Speaker Abstracts
11
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.