MicroFRET™ - Quantitative FRET Imaging with Calibration Standards

A growing number of laboratories that employ imaging technology for drug discovery, cell biology and high-throughput screening are seeking to exploit the phenomenon of fluorescence resonance energy transfer (FRET) as a tool for measuring distances at the molecular scale. Although several different FRET imaging techniques are available, many researchers would prefer to obtain quantitative data without resorting to photobleaching the sample or using expensive and difficult-to-operate equipment.

MicroFRET technology integrates optics, software algorithms, molecular reagents and calibrants. Selected FRET pairs, including fluorescent proteins and dyes, are used as FRET calibration standards, which can be applied in combination with the software to deconvolute donor and acceptor signals from FRET signals, thereby eliminating the non-FRET fluorescence emission from the FRET channel.

Elimination of any cross-talk among the channels and spectral overlap (‘bleed-through’) from the donor and acceptor channels into the FRET channel makes it possible to generate a color-corrected RGB image in which the donor pixels are colored blue, the acceptor pixels are colored green, and the FRET pixels are colored red (see figure, below). This data can be used to obtain detailed information about the interactions of components in the sample at a scale which is smaller than the optical resolution limit.

MicroFRET analysis of a mixed population of three bead types with bound derivatives of the Green Fluorescent Protein (GFP): pure donor (blue), pure acceptor (green), and donor + acceptor, resulting in FRET (red).

A more complete description of MicroFRET is given in an HTML manuscript on this site, and a reprint of the published manuscript is available to browsers which support MS Word or PDF.

MicroFRET patents are issued and pending.

 

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