Commentary: Assessing FRET using spectral techniques

Abstract

The manuscript “Assessing FRET using spectral techniques” by Leavesley et al. (page 898 in this issue) addresses Förster resonance energy transfer (FRET) between organic molecules due to an interaction of optical transition dipole moments. Although occurring in nature for more than a billion of years, a theoretical description of this mechanism has only been given in 1948 1. Since that time energy transfer spectroscopy has been used to probe molecular distances and mechanisms in the nanometre range 2-4, profiting from the fact that the energy transfer rate depends on the sixth power of molecular distance (kET ∼ r−6) and is, therefore, a very sensitive parameter. FRET experiments gained considerable importance, when in the 1990s green fluorescent protein [GFP, naturally occurring in the jellyfish Aequorea Victoria, 5] and its mutants could be cloned and fused with almost any protein of a cell. So, nonradiative energy transfer from cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP) or from GFP to red fluorescent protein was used to probe changes of molecular conformation, for example, upon binding of calcium 6, or interactions between adjacent molecules, for example, protein−protein interactions playing an important role in regulation of apoptosis 7 or in pathogenesis of M. Alzheimer and further neurodegenerative diseases 8, 9. Due to these experimental restrictions (and pronounced costs) of time-resolving FRET experiments, spectral techniques and algorithms upon stationary optical excitation still play an important role, as outlined in the manuscript by Leavesley et al. (page 898 in this issue). Here, the authors used various techniques and algorithms based on up to three filter sets (e.g., for measurement of FRET efficiency at variable donor concentration) as well as spectral unmixing. Using a probe, where CFP and YFP are linked by a cAMP binding protein, they measured FRET efficiency in cultivated cells as a function of cAMP concentration. Although a two-filter set approach was already able to describe FRET efficiency with rather low coefficients of variation, spectrally resolved experiments provided improved results. The authors concluded that hyperspectral confocal microscopy in combination with linear unmixing, cell segmentation, and quantitative image analysis was appropriate for FRET experiments in single cells. I would like to share this opinion, emphasizing that in an ideal case spectral and time-resolving FRET experiments might be combined. This holds in particular, if acceptor fluorescence is low 8, or if donor and acceptor fluorescence are spectrally indiscernible [Homo FRET, 11]. During the last years resolution in optical microscopy has been improved considerably. Starting from a value around 200 nm, as resulting from Abbé's diffraction theory, values of 10−30 nm were meanwhile obtained by super-localization microscopy based on single molecule detection 12, 13 or by stimulated emission depletion microscopy [STED, 14]. Resolution in FRET experiments still goes below these values and permits to measure molecular distances of 5 nm and less. In addition, high irradiance as needed for STED (and to some extent also for single molecule detection) and risking severe cell damage can be avoided in FRET experiments. Furthermore, FRET is not restricted to microscopy and not limited to adherent samples, but can be combined with any other method, for example, flow cytometry 15.