Lifetime imaging of FRET between red fluorescent proteins

Abstract

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.

Modeling Structures and Spectra of Fluorescent Proteins in the Coordinate-Locking Cluster Approach: Application to the Photoswitchable Protein AsFP595

“…Also, KFP is considered a convenient acceptor partner in the Förster resonance energy transfer (FRET) pair with brightly fluorescent red protein TagRFP. Employing FRET between these two proteins connected by an appropriate peptide linker opens new perspectives for monitoring biological and physiological mechanisms and for creating novel diagnostic tools [45,46]. Beyond promising practical applications, studies of asFP595 are of considerable importance, due to challenging puzzles in its photophysical behavior.…”

Genetically Encoded Far-Red Fluorescent Sensors for Caspase-3 Activity

“…Such multiparameter imaging is very important for deciphering the true order of events and the correlations between them since individual cells behave non-synchronously. Compared to red caspase-3 sensors RACS3 (9) and TagRFP-23-KFP (26), mKate2-DEVD-iRFP possesses strongly red-shifted spectra for both donor and acceptor. Potentially, this makes it possible to use mKate2-DEVD-iRFP together with orange or even red fluorescent proteins [see examples of multicolor labeling with mKate2 and mOrange2 (27) or TagRFP (28)], which are clearly incompatible with RACS3 and TagRFP-23-KFP.…”

An In Vivo Spectral Multiplexing Approach for the Cooperative Imaging of Different Disease-Related Biomarkers with Near-Infrared Fluorescent Förster Resonance Energy Transfer Probes

“…This is by far a simpler strategy than using systems in which chromophore-modified (fused) proteins are to be incorporated into cells in order to detect specific molecule interactions (7)(8)(9)30,31). Even though this in vivo approach represents a proof of concept, labeling of macrophages via FRET is expected to work also in other disease models related to spontaneous inflammatory processes, such as rheumatoid arthritis.…”

“…FRET is a phenomenon that takes place when 2 different chromophores (donor and acceptor) with overlapping emission/absorption spectra undergo long-range dipole-dipole coupling (3,4). Several interesting approaches have been reported from microscopic analysis in vitro (5)(6)(7)(8) and ultimately also in vivo (9). In the latter case, mutants of green fluorescent protein (10,11) with varying spectral properties have been used together with recombinant techniques to introduce those fused proteins containing the respective FRET donor and acceptor chromophores into cell systems.…”