KEYWORDS: Luminescence, Fluorescence resonance energy transfer, Microfluidics, Photons, Signal detection, Proteins, Green fluorescent protein, Cancer, Signal processing, Fluorescence lifetime imaging
Cancer studies require a thorough understanding of how human gene expressions and DNA modifications are translated
at the proteome level. In order to unravel the large and complex interactions between proteins, we have developed a
compact lifetime-based flow cytometer, utilising a commercial microfluidic chip, to screen large non-adherent cell
populations. Fluorescent signals from cells are detected using time correlated single photon counting (TCSPC) in the
burst integrated fluorescence lifetime (BIFL) mode and used to determine the lifetime of each cell. Initially, the system
was tested using 10 μm highly fluorescent beads to determine optical throughput and detection efficiency. The system
was validated with 293T monkey kidney adenocarcinoma cell line transiently transfected with a FRET standard,
consisting of eGPF and mRFP1 fluorescent proteins linked by a19 amino-acid chain. Analysis software was developed to
process detected signals in BIFL mode and chronologically save the transient burst data for each cell in a multi-dimensional
image file.
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