Method of Simultaneous Frequency-Sweeping Lifetime Measurements on Multiple Excitation Wavelengths

A new lifetime imaging system that can fully measure the FRET network between five fluorescent labels in a single image scan has been developed at the University of Arizona. This technology is capable of performing hyper-dimensional lifetime imaging on multiple excitation wavelengths simultaneously, allowing for previously not achievable FRET imaging of cellular processes. The system also is cost-effective as no pulsed lasers or time-gating mechanisms are required for collecting images, and the design can easily be configured for any fluorophore combination.
 

Background:

Real-time studying of signaling pathways in cells is limited by our inability to perform Föster resonant energy transfer (FRET) imaging among four or more fluorescent labels. Although two-label FRET imaging is routinely used in research, imaging FRET between more than two fluorophores remains highly challenging. Lifetime imaging can distinguish paired (quenched) donor fluorophores from unpaired (unquenched) donors, measure the absolute FRET efficiency, and fully quantify the FRET process. A fast acquisition FRET lifetime imaging system capable of measuring lifetime data from simultaneous multi-wavelength excitation could monitor complex FRET networks in live cell processes. Such a system could be used to help understand mechanisms of genetic disorders, cancers, and more.
 

Advantages:

• Comparatively short acquisition time
• Simultaneous FRET lifetime measurements for up to five fluorophores
• Relatively low cost and good configurability for different fluorophore combinations

Applications:

• Dynamic imaging of signaling pathways in cells
• Biomedical imaging of protein interactions for use in studying genetic disorders, cancers, etc.

Status: issued U.S. patent #9,217,710