In FRET applications, spectral imaging can be considered a variation of the sensitized emission technique that relies on excitation of the donor alone, followed by acquisition of the entire emission spectrum of both the donor and acceptor fluorescence instead of capturing data in two independent channels. Until the introduction of laser scanning confocal microscopes designed for spectral imaging, the technique was largely limited to spectroscopy experiments using cuvettes and purified fluorophores. Spectral imaging FRET assumes that gathering of the entire fluorescence spectrum will enable overlapping spectral profiles to be separated according to the distinct shapes of the spectra rather than simply monitoring emission intensity in a limited bandwidth region using a filter.
Molecular Imaging, Chapter 10 Academic Press, Burlington, MA, 336 pages (2005). One of the best review articles on FRET analysis with spectral imaging. The investigators review the basics of spectral imaging and discuss acceptor photobleaching techniques, elimination of signal bleed-through, image collection parameters, and potential problems and pitfalls.
Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Letters 531: 245-249 (2002). One of the original papers coupling spectral imaging and linear unmixing with FRET using fluorescent proteins. Using a long Stokes shift donor, FRET efficiency is determined for variants of GFP and EYFP.
Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions. Journal of Microscopy 228: 139-152 (2007). An algorithm is described in this report that measures and removes the contaminating acceptor signal bleed-through signal from spectral imaging data in FRET specimens. The method is then used to detect the dimerization of a transcription factor in living cells.
Applying spectral fingerprinting to the analysis of FRET images. Microscopy Research and Technique 64: 185-195 (2004). Described in this research report is the theoretical analysis of how spectral fingerprinting can be applied to separate fluorescence of FRET pairs from that of unpaired donors and acceptors and how to select imaging parameters to optimize the signal-to-noise ratio. The results are compared to the expected resolution of traditional FRET measurements.
Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching. Journal of Microscopy 231: 97-104 (2008). The authors describe the adaptation of commercially available software (ZEISS) for use with acceptor photobleaching FRET using EGFP and EYFP as a FRET pair. To confirm success of the methodology, two endonuclease constituents of the nucleotide excision repair system were used to demonstrate FRET.
Application of Spectral Imaging Microscopy in Cytomics and Fluorescence Resonance Energy Transfer (FRET) Analysis. Cytometry A 59A: 172-181 (2004). A general review article with research highlights on spectral imaging and linear unmixing FRET with filters to demonstrate superiority of the technique with respect to detection accuracy when compared to traditional methods. The article contains helpful information on the removal of autofluorescence.
Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing. Journal of Microscopy 215: 162-173 (2004). The authors couple spectral imaging with acceptor photobleaching to examine FRET in gap junctions using ECFP and EYFP fusions to connexin Cx26. They conclude that spectral imaging produces results similar to sensitized emission and performs well in cell populations with variable plasmid expression levels.
Megias, D., Marrero, R., Del Peso, B., Garcia, M., Bravo-Cordero, J., Garcia-Grande, A., Santos, A. and Montoya, M.
Novel Lambda FRET Spectral Confocal Microscopy Imaging Method. Microscopy Research and Technique 72: 1-11 (2009). Introducing a technique known as lambda FRET, the authors describe a novel off-line precalibration procedure to eliminate spectral bleed-through based on the acquisition of reference reflection images.
Quantitative Multiphoton Spectral Imaging and Its Use for Measuring Resonance Energy Transfer. Biophysical Journal 89: 2736-2749 (2005). The authors introduce an unmixing algorithm that incorporates resonance energy transfer variables for confocal and multiphoton imaging. Termed sRET, the method was confirmed using Cerulean and Venus standards with defined FRET efficiencies.
Correlated Fluorescence Lifetime and Spectral Measurements in Living Cells. Microscopy Research and Technique 70: 85-94 (2007). An excellent research report describing the complementary aspects of correlated fluorescence lifetime and spectral imaging for FRET analysis in live cells. The authors present the SLiM-SPRC160, an acquisition system for simultaneous measurements.