High-Contrast Fluorescence Imaging in Fixed and Living Cells Using Optimized Optical Switches

We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C₁₂-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.     

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.     

Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes

GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image acquisition and processing are also explained. An optimal FRET probe should have (i) a wide dynamic range (which means a high sensitivity), (ii) a high fluorescence intensity, (iii) target specificity, and (iv) a minimal perturbation to endogenous signaling cascades. Although an improvement of FRET probes should be executed in a trial-and-error manner, practical tips for optimization are provided here. In addition, we illustrate some applications of FRET probes for neuronal cells, which are composed of diverse subcellular compartments with different functions; thus, tools to decipher the dynamics of GTPase activity in each compartment have long been desired.     

Single- and dual-parameter FRET kinase probes based on pleckstrin

Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create diverse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d; microscopy and image processing take 2 h.     

Fluorescent kabiramides: new probes to quantify actin in vitro and in vivo

We present the design, synthesis, and biochemical and spectroscopic characterization of five functional fluorescent conjugates of kabiramide C (KabC), a small molecule biomimetic of gelsolin. The tetramethylrhodamine (TMR), rhodol green (RG), IC5, dapoxyl (DAP), and fluorescein diester (FDE) conjugates of KabC bind specifically to actin at the barbed end in a 1:1 complex. These probes are shown to function in an indistinguishable manner to the unmodified KabC. Various modalities of the fluorescence emission of these KabC probes, including fluorescence anisotropy and fluorescence resonance energy transfer, are used for the development of assays for the rapid determination of G-actin concentration in solution. The TMR-KabC and FDE-KabC probes are cell permeable and provide unique imaging information on the distribution and dynamics of actin filament within living cells.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

Effects on the conformation of FVIIa by sTF and Ca binding: Studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5 Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044–1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.It is known from amidolytic activity measurements that Ca²⁺ binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca²⁺ binding. Interestingly, this Ca²⁺-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Switch on or switch off: an optical DNA sensor based on poly(p-phenylenevinylene) grafted magnetic beads

There has been an enormous demand for commercial label-free DNA sensors in a diverse range of fields including pre-emptive medicine, diagnostics, environmental monitoring, and food industry. Addressing the need for sensitive, selective and facile DNA sensors, we demonstrate a novel switch on/off sensor design that utilizes sandwich hybridization between photoluminescent anionic conjugated polyelectrolyte (CPE) bound captureprobe coated onto magnetic beads, target and the signaling probe. The hybridization-readout in our sensor was monitored by either fluorescence resonance energy transfer (FRET, switch-on) or superquenching (switch-off) depending on the type of signaling probe used. Moreover recent designs that utilize beads for sensing DNA have been limited towards using electrostatic interactions or intercalation of dyes to observe FRET. To our knowledge this is the first report of a switch on/off sensor utilizing either FRET or superquenching thus providing flexibility for future development of such rapid, facile and sensitive DNA sensors. The FRET-based sensor was investigated by optimizing the reaction parameters and selectivity. A low detection limit of 240 fmol in 2 mL of SSC buffer was achieved.     

Effects on the conformation of FVIIa by sTF and Ca²⁺ binding: studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5? for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4? upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.     

Measuring protein conformational changes by FRET/LRET

Fluorescence resonance energy transfer (FRET) provides a unique means of measuring interatomic distances in biological molecules in real time. Recent advances have been made in the application of this technique to studies of conformational changes in proteins. New ways of introducing fluorescence probes into proteins, newly developed fluorescence probes, and progress in the technologies for fluorescence signal detection have greatly expanded the range of applications of FRET. In particular, studies of conformational changes in proteins at a single molecule level and in the native in vivo context of a living cell are now possible.     

Linear Approaches to Intramolecular F?rster Resonance Energy Transfer Probe Measurements for Quantitative Modeling

Numerous unimolecular, genetically-encoded Förster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R_alt) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R_alt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.     

Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells

Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)(+)-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25nM to 0.5μM) of poly(A)-RNA, as well as for determination of K(d) values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed.