Automated single-molecule imaging in living cells

Abstract

An automated single-molecule imaging system developed for live-cell analyses based on artificial intelligence-assisted microscopy is presented. All significant procedures, i.e., searching for cells suitable for observation, detecting in-focus positions, and performing image acquisition and single-molecule tracking, are fully automated, and numerous highly accurate, efficient, and reproducible single-molecule imaging experiments in living cells can be performed. Here, the apparatus is applied for single-molecule imaging and analysis of epidermal growth factor receptors (EGFRs) in 1600 cells in a 96-well plate within 1 day. Changes in the lateral mobility of EGFRs on the plasma membrane in response to various ligands and drug concentrations are clearly detected in individual cells, and several dynamic and pharmacological parameters are determined, including the diffusion coefficient, oligomer size, and half-maximal effective concentration (EC50). Automated single-molecule imaging for systematic cell signaling analyses is feasible and can be applied to single-molecule screening, thus extensively contributing to biological and pharmacological research.

Needle Biopsy Adequacy in the Era of Precision Medicine and Value-Based Health Care

“…Single cell analysis is an emerging analytical technique to characterize properties of rare cells, to analyze cell expression heterogeneity, 59,60 and even to characterize properties of a single molecule class within a cell. 61 For single-cell analysis, accessing and extracting single cells with minimal perturbation of native expression profiles remains challenging. 62…”

Bridging the Nanoscopy-Immunology Gap

“…If super-resolution microscopy is to be used for population based imaging, the number of cells imaged, and their diversity must be increased. To achieve this, researchers have been making efforts toward increasing the throughput of an average super-resolution experiment by automating the process [47,48], and by using flat field illumination to increase the number of cells collectable in each image [49,50].…”

“…Automated super-resolution imaging and single particle tracking has been performed by Masato Yasui of the Ueda lab in Osaka [47]. His system uses fast focusing based on pixel intensity at the iris to select the correct focal plane, then machine learning to find and image cells with a given morphology and fluorescent signal.…”

Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR

“…The activation-dependent decrease of the diffusion coefficient is also commonly observed with the epidermal growth factor receptor (EGFR) [5] and TRPV1 channel [6]. Therefore, the diffusion coefficient is a key index reflecting the functional states of various membrane receptors.…”

“…This situation would make it difficult for trainees to perform singlemolecule image analysis. Furthermore, the sample throughput of the SMI has been increased due to the development of the automated SMI system [5,18], and the image analysis has become the ratelimiting phase of the experiments.…”