Automation and High-Throughput Technologies

Automated Fluorescence Lifetime Imaging Assays in 2-D and 3-D Cell Culture

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  Sunil Kumar, MSc, MRes, PhD

  Research Associate
  Imperial College London

We present an open source, automated multiwell plate fluorescence lifetime imaging (FLIM) high content analysis platform to assay protein interactions and read out genetically-expressed biosensors utilizing Förster resonant energy transfer (FRET). This instrument provides automated acquisition of optically sectioned fluorescence lifetime images utilising a Nipkow spinning disc scanner and wide-field time-gated imaging and we have demonstrated its application to 2-D and 3-D cell cultures.
In 2-D cell culture we demonstrate the potential to assay protein-protein interactions in fixed or live cells and measure the kD of interactions between the MST1 kinase and members of the RASSF protein family, which are of importance for apoptosis via the Hippo signaling pathway. This assay utilises GFP and mCherry-labelled proteins and combines quantitative fluorescence intensity measurements with FLIM data globally fitted to a double exponential decay profile. Recognizing that this model does not correspond to the underlying behavior of fluorescent proteins undergoing FRET, owing to their slow rotational correlation time contradicting the assumption of FRET averaged over rapidly varying orientations of fluorophore dipoles, we have developed a new tool to analyse FLIM FRET data based on the assumption of static randomly orientated fluorophore dipoles that can be used to correct conventional FLIM FRET data.
To translate our FLIM FRET HCA platform to 3-D cell culture, we have applied the automated optically sectioning multiwell plate FLIM microscope to tumour spheroids expressing FRET biosensors. In particular, we have applied FLIM to cells expressing T2AMPKAR (a version of the AMPKAR FRET biosensor modified to replace CFP with mTq2FP) which can be used to map the activation of AMP activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. We first validated this FRET biosensor in HEK293T cells by obtaining the dose response curve for known activators of AMPK in an automated 2-D FLIM FRET assay and then expressed T2AMPKAR in tumour spheroids and showed that we obtain equivalent dose response curves using the same multiwell plate optically sectioned FLIM platform. We have also studied the FLIM FRET readout of T2AMPKAR with multiphoton microscopy.
The software for automated FLIM data acquisition is written in Micromanager and our global analysis software tool, FLIMfit, is written in MATLAB. Both are openly available and can be accessed at http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/