An Automated Live‐Cell Microscopy System for Drug Profiling and Screening

Abstract

Time‐lapse microscopy combined with fluorescent biosensors is a powerful tool to quantitatively measure cellular signaling dynamics and observe cellular morphology with high spatial and temporal resolution. Unfortunately, the collection of these data can be time intensive due to the serial collection of data and manual addition of small volumes of stimulants. While perfusion systems for microscopes can automate the delivery of drugs, they often require a relatively large amount of drug and may limit the number of different drugs that can be loaded at a given time. To address these issues, we have developed a highly customizable platform that integrates an open‐source liquid handling robot with a motorized, inverted, epifluorescence microscope that is capable of automating multi‐well, time‐course microscopy and small volume drug addition.This system has been tested and validated using fluorescent biosensors A‐Kinase Activity Reporter (AKAR) and Indicator for cAMP Using Epac (ICUE), for Protein Kinase A, PKA, and cyclic Adenosine monophosphate, cAMP, respectively where PKA and cAMP responses from a large number of individual, living cells can be faithfully recorded. By using an 8‐channel micropipette and acquiring images from 8 different wells in parallel we were able to increase the throughput of collecting these data. Thus, from a single 96 well plate we were able to collect multiple repeats of a detailed dose response curve of the AKAR4 response to isoproterenol, a β‐adrenergic receptor agonist, in HEK293T cells. While the aggregated cell responses conformed very nicely to an ideal dose response curve, there was a significant cell‐to‐cell variability in the individual responses at intermediate doses, which highlights the importance of increasing the throughput of these single cell measurements. In addition to increasing the number of repeated measures, this system increases the number of different stimulants that can be tested. As a proof of concept, we have used these fluorescent biosensors to functionally profile a panel of G‐Protein‐Coupled Receptor, GPCR, agonists and antagonists and evaluate the differences in responses across cell types. Additionally, we are using this system to screen a library of marine natural products for possible GPCR agonists or antagonists using these fluorescent biosensors as a functional readout of GPCR responsiveness. These data have shown that this system has expanded the usefulness of fluorescent biosensors into the realm of drug profiling and screening that would not be plausible with the manual method of acquisition. Furthermore, the open‐source and flexible nature of this tool makes it a resource for increasing throughput of fluorescence microscopy that is accessible to a diverse set of labs and applications.Support or Funding InformationThis work has been funded by the following NIH grants: F32 GM120798‐02, R35 CA197622, and R01 DK073368This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.