Picosecond-resolution fluorescence lifetime imaging microscopy: a useful tool for sensing molecular interactions in vivo via FRET

Wei Zhong,Karl A Merrick,Mary-Ann Mycek,Mei Wu,Ching-Wei Chang,Sofia D Merajver

doi:10.1364/oe.15.018220

Abstract

Fluorescence lifetime imaging microscopy (FLIM) provides a promising, robust method of detecting molecular interaction not not nots in vivo via fluorescence/Förster resonance energy transfer (FRET), by monitoring the variation of donor fluorescence lifetime, which is insensitive to many artifacts influencing convential intensity-based measurements, e.g. fluorophore concentration, photobleaching, and spectral bleed-through. As proof of principle, we demonstrate the capability of a novel picosecond-resolution FLIM system to detect molecular interactions in a well-established FRET assay. We then apply the FLIM system to detect the molecular interaction of a transforming oncogene RhoC with a binding partner RhoGDIgamma in vivo, which is critical to understand and interfere with Rho signaling for cancer therapeutics.

Highlights

The ability to rapidly characterize molecular function in vivo would be a fundamental advance in biology and medicine
An excitation pulse illuminates a sample and an image of the fluorescence emission is acquired by an intensified charge-coupled device (ICCD) camera at a controllable intensifier gate delay tG, with emission intensities integrated during the gate width Δt
Because of the wide tunability of the dye laser, the Fluorescence lifetime imaging microscopy (FLIM) system could be adapted to studies of enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) pairs in living cells

Summary

Introduction

The ability to rapidly characterize molecular function in vivo would be a fundamental advance in biology and medicine. Traditional biophysical or biochemical methods, such as affinity chromatography or co-immunoprecipitation, and more recently, two-hybrid and phage-display methods have been used to detect molecular interactions in vitro [1,2,3,4]. Biologically important molecules that exist in more than one activation state are hard to isolate from the complex in vivo system while preserving the integrity of their activation cycle. One such molecule, RhoC, has been found to be a transforming oncogene. The biophysical mechanisms for activation and inhibition of this oncogene (including detailed molecular associations and cellular localization) are not well understood in part due to the limitations of traditional biophysical and biochemical methods

Methods

Results

Conclusion