Additional correction for energy transfer efficiency calculation in filter-based Förster resonance energy transfer microscopy for more accurate results

Details Additional correction for energy transfer efficiency calculation in filter-based Förster resonance energy transfer microscopy for more accurate results Additional correction for energy transfer efficiency calculation in filter-based Förster resonance energy transfer microscopy for more accurate results

Mar 2010

adsabs.harvard.edu/cgi-bin/nph-data_query?bibcode=2010jbo....15b0513s&link_type=abstract

Journal of Journal of Biomedical Optics, Volume 15, Issue 2, pp. 020513-020513-3 (2010).

Physics

Optics

1

Band-Pass Filters, Biological Techniques, Bio-Optics, Fluorescence, Molecular Biophysics, Optical Filters, Optical Microscopy, Proteins, Biological Techniques And Instrumentation, Biomedical Engineering, Optical Microscopy, Proteins, Conventional Optical Microscopes, Filters, Zone Plates, And Polarizers

Scientific paper

Förster resonance energy transfer (FRET) microscopy is commonly used to monitor protein interactions with filter-based imaging systems, which require spectral bleedthrough (or cross talk) correction to accurately measure energy transfer efficiency (E). The double-label (donor+acceptor) specimen is excited with the donor wavelength, the acceptor emission provided the uncorrected FRET signal and the donor emission (the donor channel) represents the quenched donor (qD), the basis for the E calculation. Our results indicate this is not the most accurate determination of the quenched donor signal as it fails to consider the donor spectral bleedthrough (DSBT) signals in the qD for the E calculation, which our new model addresses, leading to a more accurate E result. This refinement improves E comparisons made with lifetime and spectral FRET imaging microscopy as shown here using several genetic (FRET standard) constructs, where cerulean and venus fluorescent proteins are tethered by different amino acid linkers.

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