No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
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.
... FLIM allows discrimination of two different red FPs in subcutaneous mouse models. 52 Fluorescence lifetime imaging-Forster resonance energy transfer (FLIM-FRET) techniques are based on the measurement of the fluorescence lifetime of the donor in the FRET pair. This method overcomes the problems of fluctuation of fluorophore concentration in biological samples and does not require a donor calibration procedure. ...
... 61 Earlier we used FLIM-FRET for detection of caspase-3 activation in tumor cells in vitro. 52,53 This paper describes single photon FLIM-FRET of a genetically encoded sensor for caspase-3 imaging in vivo in mouse tumor xenografts generated from A549 and HEp-2 cells. We demonstrate that this system allows evaluation of chemotherapeutic intervention efficiency toward tumor cells within an animal model. ...
... Because KFP is almost nonfluorescent, 62 there is no spectral crosstalk of donor and acceptor, and it is The DEVD tetrapeptide is embedded in the linker and specifically recognized by caspase-3 and cleaved, leading to the increase of TagRFP fluorescence intensity and lifetime. 52 The use of the latter parameter is more convenient because it is independent of such factors as donor photobleaching and concentration of the sensor. Despite the tetrameric structure of TagRFP-23-KFP sensor, the DEVD site in the linker is located far enough from the barrels of TagRFP and KFP to be cleaved by caspase-3 without steric hindrances. ...
Effectiveness of extended depth of field microscopy (EDFM) implementation with wavefront encoding methods is reduced by depth-induced spherical aberration (SA) due to reliance of this approach on a defined point spread function (PSF). Evaluation of the engineered PSF's robustness to SA, when a specific phase mask design is used, is presented in terms of the final restored image quality. Synthetic intermediate images were generated using selected generalized cubic and cubic phase mask designs. Experimental intermediate images were acquired using the same phase mask designs projected from a liquid crystal spatial light modulator. Intermediate images were restored using the penalized space-invariant expectation maximization and the regularized linear least squares algorithms. In the presence of depth-induced SA, systems characterized by radially symmetric PSFs, coupled with model-based computational methods, achieve microscope imaging performance with fewer deviations in structural fidelity (e.g., artifacts) in simulation and experiment and 50% more accurate positioning of 1-μm beads at 10-μm depth in simulation than those with radially asymmetric PSFs. Despite a drop in the signal-to-noise ratio after processing, EDFM is shown to achieve the conventional resolution limit when a model-based reconstruction algorithm with appropriate regularization is used. These trends are also found in images of fixed fluorescently labeled brine shrimp, not adjacent to the coverslip, and fluorescently labeled mitochondria in live cells.
... FLIM allows discrimination of two different red FPs in subcutaneous mouse models. 52 Fluorescence lifetime imaging-Forster resonance energy transfer (FLIM-FRET) techniques are based on the measurement of the fluorescence lifetime of the donor in the FRET pair. This method overcomes the problems of fluctuation of fluorophore concentration in biological samples and does not require a donor calibration procedure. ...
... 61 Earlier we used FLIM-FRET for detection of caspase-3 activation in tumor cells in vitro. 52,53 This paper describes single photon FLIM-FRET of a genetically encoded sensor for caspase-3 imaging in vivo in mouse tumor xenografts generated from A549 and HEp-2 cells. We demonstrate that this system allows evaluation of chemotherapeutic intervention efficiency toward tumor cells within an animal model. ...
... Because KFP is almost nonfluorescent, 62 there is no spectral crosstalk of donor and acceptor, and it is The DEVD tetrapeptide is embedded in the linker and specifically recognized by caspase-3 and cleaved, leading to the increase of TagRFP fluorescence intensity and lifetime. 52 The use of the latter parameter is more convenient because it is independent of such factors as donor photobleaching and concentration of the sensor. Despite the tetrameric structure of TagRFP-23-KFP sensor, the DEVD site in the linker is located far enough from the barrels of TagRFP and KFP to be cleaved by caspase-3 without steric hindrances. ...
Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.
... 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. ...
... Using the edema model, we have shown, for what is to our knowledge the first time, that FRET is possible when a contrast agent cocktail is administered into a nonengineered living organism. 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. ...
In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Förster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies.
We coupled the fluorophores DY-682 (donor; absorption [abs]/emission [em], 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system with multispectral analysis features.
When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization of the DY-682-IgG-DY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice.
Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.
... We note that, although FLIM and FRET are well established for cell microscopy, FLIM has only recently been demonstrated in live mouse models, implemented with tomography to image dye phantoms and subcutaneous tumors targeted with a fluorescent marker [7] or expressing a fluorescent protein [8]. To date, intensity-based FRET tomography [9] and FLIM FRET have only been applied to mice ex vivo [8]. ...
... We note that, although FLIM and FRET are well established for cell microscopy, FLIM has only recently been demonstrated in live mouse models, implemented with tomography to image dye phantoms and subcutaneous tumors targeted with a fluorescent marker [7] or expressing a fluorescent protein [8]. To date, intensity-based FRET tomography [9] and FLIM FRET have only been applied to mice ex vivo [8]. We report here a tomographic approach to monitor in vivo FLIM FRET readouts and demonstrate for the first time the reconstruction of the lifetime and quantum yield of a genetically expressed FRET probe measured in vivo. ...
Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.
... Athymic mice (nu/nu, n=6-7) were implanted with subcutaneous epithelioid human carcinoma cells (HEp2 or A549) that formed tumor xenografts expressing Tag RFP, a far-red cell marker protein previously used for sensor engineering and in tumor detection. 7,8 TagRFP has an emission maximum of 584 nm 9 and its fluorescence can be imaged in vivo at 2-3 weeks after tumor inoculation into the flank of the animals. 10 Initial observation using a whole-body imaging system (FI imaging) identified red fluorescent tumor nodules, and their signal-to-background (conspicuity) underwent a substantial increase after OC application ( Figure 1B) with both 1 M GB and 0.7M GB OC mixture. ...
... The caspase activity sensor was based on TagRFP as a f luorescence donor and chromoprotein KFP (kindling f luorescent protein) as an acceptor. In vivo caspase-3 activation in response to the antitumor therapy was monitored noninvasively over a long period of time (about 30 days) [170][171][172]. ...
One of the latest methods in modern molecular biology is labeling genomic loci in living cells using fluorescently labeled Cas protein. The NIH Foundation has made the mapping of the 4D nucleome (the three-dimensional nucleome on a timescale) a priority in the studies aimed to improve our understanding of chromatin organization. Fluorescent methods based on CRISPR-Cas are a significant step forward in visualization of genomic loci in living cells. This approach can be used for studying epigenetics, cell cycle, cellular response to external stimuli, rearrangements during malignant cell transformation, such as chromosomal translocations or damage, as well as for genome editing. In this review, we focused on the application of CRISPR-Cas fluorescence technologies as components of multimodal imaging methods for in vivo mapping of chromosomal loci, in particular, attribution of fluorescence signal to morphological and anatomical structures in a living organism. The review discusses the approaches to the highly sensitive, high-precision labeling of CRISPR-Cas components, delivery of genetically engineered constructs into cells and tissues, and promising methods for molecular imaging.
... Fluorescence lifetime imaging requires time-resolved measurements, which are known to be done either in time or in frequency domain. Both fluorescence lifetime imaging microscopy 8,[24][25][26][27] and FMT prefer the time-domain measurement techniques to the frequency-domain ones because in the frequency domain, modulation of light sources is technologically limited by frequencies about 2 GHz. 28 This makes it nearly impossible to measure the entire Fourier spectrum and hence collect the frequency domain data that are equivalent to time domain data in terms of information content. ...
Significance: Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons. Aim: The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry. Approach: The sensitivity functions were simulated with a Monte Carlo method. A compressed sensing like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method. Results: 3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object. Conclusions: Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.
... Fluorescence lifetime imaging requires time-resolved measurements, which are known to be done either in time or in frequency domain. Both fluorescence lifetime imaging microscopy 8,[24][25][26][27] and FMT prefer the time-domain measurement techniques to the frequency-domain ones because in the frequency domain, modulation of light sources is technologically limited by frequencies about 2 GHz. 28 This makes it nearly impossible to measure the entire Fourier spectrum and hence collect the frequency domain data that are equivalent to time domain data in terms of information content. ...
Significance:
Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons.
Aim:
The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry.
Approach:
The sensitivity functions were simulated with a Monte Carlo method. A compressed-sensing-like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method.
Results:
3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object.
Conclusions:
Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.
... 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]. ...
An interest in the fluorescent protein asFP595 is due to unexplained puzzles in its photophysical behavior. We report the results of calculations of structures, absorption, and emission bands in asFP595 by considering model molecular clusters in the coordinate-locking scheme. Both trans and cis conformations of the anionic chromophore are considered. Equilibrium geometry coordinates on the ground potential energy surface were optimized in the density functional theory approaches by considering both large- and reduced-size clusters. The cluster size was reduced to locate positions of the minimum energy points on the excited-state potential surface by using the configuration interaction singles approach. Vertical excitation energies and oscillator strengths were computed by using the ZINDO method. We show that consideration of large clusters mimicking the protein-containing pocket is an essential issue to calculate positions of absorption and emission bands with the accuracy compatible to experiments.
... For this work, we concentrate on the application of FRET to the colored protein systems of the green fluorescent protein (GFP) family, well-known molecular markers used in living cells (7)(8)(9). More specifically, we are interested in theoretical evaluation of the FRET parameters in these systems, which can be used when constructing novel sensors (10)(11)(12), as well as in the interpretation of complicated spectral features of fluorescent proteins. ...
The orientation factor κ2, one of the key parameters defining Förster resonance energy transfer efficiency, is determined by the transition dipole moment orientations of the donor and acceptor species. Using the results of quantum chemical and quantum mechanical/molecular mechanical calculations for the chromophore-containing pockets in selected colored proteins of the green fluorescent protein family, we derived transition dipole moments corresponding to the S0,min → S1 excitation for green fluorescent protein, red fluorescent protein (TagRFP), and kindling fluorescent protein, and the S1,min → S0 emission for TagRFP. These data allowed us to estimate κ2 values for the TagRFP-linker-kindling fluorescent protein tetrameric complex required for constructing novel sensors.
... Otherwise, the SFDT inverse problem cannot be properly solved. There are several techniques for autofluorescence removal that are widely employed in the fluorescence imaging: the spectral unmixing method [18,19], which uses the difference in excitation or emission spectra for fluorescence and autofluorescence, and fluorescence lifetime measurements made by the time-domain [20][21][22] or frequency-domain [22,23] techniques. In application to SFDT, both autofluorescence removal techniques are time consuming and greatly increase the complexity of the system and instrumentation cost. ...
The dispersion of biotissue optical properties results in distortion of the spectrum of radiation during propagation through biotissues. This phenomenon can be used for solving the inverse problem in fluorescence diffuse tomography. We have developed the spectrally resolved fluorescence diffuse tomography (SFDT) technique, which allows reconstruction of the spatial distribution of the fluorophore in biotissue even in the presence of unknown autofluorescence. The experimental setup combining epi- illumination and trans-illumination imaging geometries with spectral resolution has been created. Experimental studies on tissue phantoms and small animals in vivo with the proposed SFDT technique have been conducted. The obtained results have shown high accuracy of the inverse problem solution for spatial distribution of fluorophore concentration.
... A novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity was developed [154]. Lifetime-based FRET measurements using the nonfluorescent protein KFP as an acceptor help to minimize crosstalk. ...
Fluorescence lifetime (FLT) measurements in the long-wavelength red and near-infrared (NIR) range are expected to improve the reliability and robustness of fluorescence-based detection. This review provides a summary of suitable classes of red and NIR luminescent reporters for use in FLT-based applications.
... Recently we have reported construction of a FRET pair on the basis of red proteins with practically non-fluorescent chromoprotein KFP as the acceptor [77,78]. Cleavage of the linker under the action of protease (Fig. 4) resulted in substantial increase in intensity and fluorescence lifetime [79]. Using of dark acceptors allows one to set free the major part of the spectrum and, thus, to use additional fluorescing components; this can be considered to be additional advantage of such pairs. ...
To expand the field of research in biological systems development of extra-sensitive analytical methods is highly desirable. In this review, the latest advances in technologies relying on the fluorescence resonance energy transfer between fluorescent proteins (FP's) to visualize numerous molecular processes in living cells are discussed. Variety of FP’s as well as of novel experimental techniques allows one to choose the most appropriate tools to attack concrete problems. (© 2011 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA) (© 2011 by Astro Ltd., Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA)
... Due to the strong distance dependence of the transfer efficiency, FLIM studies can reveal inter-chromophoric distances on a nanometer scale. While quantitative FRET studies via FLIM are, in principle, possible and indeed have been reported (Jares-Erijman and Jovin, 2003;Jovin et al., 2005;Schleifenbaum et al., 2009a;Rusanov et al., 2010;Grecco and Verveer, 2011), this technique is also subject to some restrictions. This is due to the fact that, in case of FRET, the quenched donor fluorescence decay at least obeys a bi-exponential decay function in which the respective amplitudes and FLT-values are coupled. ...
Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.
... On the basis of our initial assumption that BBR/BPC type Alanine zippers share similarities to Leucine zippers, we postulated parallel dimer formation of AtBPC6 (Figure 5 I), which was supported by our homology models. To validate our previous findings, we applied the powerful technique of fluorescence resonance energy transfer (FRET) combined with two-chromophore fluorescence lifetime imaging microscopy (FLIM) [56], [57]. Our custom made FLIM system consisted of a confocal sample scanning microscope (CSSM), a spectral integrating detector for measuring fluorescence intensities and a time-correlated single-photon counting board for recording fluorescence lifetime decay [58], [59]. ...
GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.
The phenomenon of fluorescence is widely used in molecular biology for studying the interaction of light with biological objects. In this article, we present an experimental investigation of the enhancement of laser-induced fluorescence of Clytia gregaria green fluorescent protein. The laser-induced fluorescence method applied in our work combines the advantages of femtosecond laser pulses and a photonic crystal cavity, with the time dependence of the fluorescence signal studied. It is shown that a green fluorescent protein solution placed in a microcavity and excited by femtosecond laser pulses leads to an increase in fluorescence on the microcavity modes, which can be estimated by two orders of magnitude. The dependences of fluorescence signal saturation on the average integrated optical pump power are demonstrated and analyzed. The results obtained are of interest for the development of potential applications of biophotonics and extension of convenient methods of laser-induced fluorescence.
A monomeric sensor, TagRFP-23-Ultramarine (TR-23-U), for effector caspase-3 was obtained. The overlap integrals of new pairs of red fluorescent protein TagRFP with four chromoproteins were calculated. The monomeric state of the Ultramarine protein and the TR-23-U sensor was confirmed by gel filtration chromatography and dynamic light scattering. Incubation with caspase-3 showed the possibility of using the new fusion protein as a FRET sensor for apoptosis detection.
Multimodality registration of optical and magnetic resonance (MR) images in the same tissue volume in vivo may be enabled by MR contrast agents with optical clearing (OC) effect. The goals of this study were to a) investigate the effects of clinical MR contrast agent gadobutrol (GB) and its combinations as potential OC agent assisting in fluorescence intensity (FI) imaging in vivo; b) evaluate MRI as a tool for imaging of topical or systemic application of GB for the purpose of OC. Subcutaneous tumor xenografts expressing red fluorescent marker protein were used as disease models. MR imaging was performed at 1T 1H MRI using T1w‐3D GRE sequences to measure time‐dependent MR signal intensity changes by ROI analysis after image segmentation. Topical application of 1.0 M or 0.7 M GB‐containing OC mixture with water and dimethyl sulfoxide (DMSO) showed similar 30‐40% increase of tumor FI during the initial 15 min. Afterwards, the OC effect of GB on FI and tumor/background FI ratio showed a decrease over time in the case of 1.0 M GB unlike 0.7 M GB mixture, which resulted in the steady increase of FI and tumor/background ratio for 15‐60 min. The use of T1w‐3D GRE MR pulse sequences showed that concentrated 1.0 M GB resulted in MR signal loss of the skin due to high magnetic susceptibility and that signal loss coincided with OC effect. Intravenous injection of 0.3 mmol GB/kg resulted in rapid but transient 40% increase of FI of the tumors. Overall, 1T MRI enabled tracking GB‐containing OC compositions on the skin surface and tumor tissue supporting the observation of time‐dependent FI increase in vivo.
Cysteine proteases, caspases, play an important role in the process of programmed cell death. In apoptosis, the cell signaling pathways overlap at executioner caspases regardless of the initial stimulus. Caspase-3 is the key one. This review considers the sensors for activity of caspase-3 and some other caspases. Over the past decade, many such sensors utilizing a variety of detection principles have been created; fluorescence and luminescence are the most common detection methods. The intracellular sensors are of particular interest; they allow visualization of the activation of caspases in living systems. Herein, we briefly describe sensors of various designs, the advantages and disadvantages of which must be taken into account when choosing a sensor for a particular experimental system.
FRET sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows the genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ2 can be obtained by methods based on the quantum theory and combined quantum mechanics / molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions - fluorescence lifetime up to milliseconds - to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.
Optical bioimaging methods have a wide range of applications in the life sciences, most notably including the molecular resolution study of subcellular structures, small animal molecular imaging, and structural and functional clinical diagnostics of tissue layers and organs. We review fluorescent microscopy, fluorescent macroscopy, optical coherence tomography, optoacoustic tomography, and optical diffuse spectroscopy and tomography from the standpoint of physical fundamentals, applications, and progress.
Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.
Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.
This article is intended to provide a brief overview of fluorescence microscopy and its application to biological samples, with a particular emphasis on fluorescence lifetime imaging (FLIM). It includes an introduction to wide-field and laser scanning confocal and multiphoton microscopy and the analysis of the fluorescence signal with respect to wavelength, lifetime and polarisation. It also reviews recent progress extending fluorescence microscopy and FLIM to super-resolved imaging, to high throughput automated microscopy and to imaging live disease models.
The detection of interactions between molecules is of great interest for the elucidation of cellular functions and mechanisms of disease. Advances in organic and inorganic chemistry and genetics facilitate the production of sophisticated fluorescing substances with defined spectral features, which enable energy transfer provided that they are in close proximity to each other. This mechanism, which was termed 'fluorescence energy transfer,' has been enthusiastically used to measure molecular interactions between genes, proteins, etc. in cells. Initially, the methodology was restricted to in vitro studies, but now it extends to the in vivo situation with the emergence of intravital microscopy devices. Researchers are also tempted to transfer the technology to imaging whole living animals noninvasively. This chapter elucidates the principle of fluorescence resonance energy transfer (FRET) and the design of FRET pairs. Intra- and intermolecular FRET applications are also discussed and different methodologies to detect FRET elucidated.
Correct identification of different fluorophores in the fluorescence lifetime imaging in vivo requires accounting for distortion of the measured fluorescent kinetics curve due to light scattering and absorption in medium. This distortion induces the difference between real and measured lifetimes of a fluorophore. We obtained analytical expression based on diffuse approximation of radiation transfer equation that allows to refine estimating the lifetime of a fluorophore. It was shown that our approach can be applied both for analytic kinetics curves obtained by diffuse approximation, Monte Carlo simulated curves and results of model experiment. Analytical and Monte Carlo simulated curves were obtained for media with different optical properties and lifetimes corresponding to those of real fluorophores. Results of numerical simulation are confirmed by the results of the model experiment.
Förster resonance energy transfer (FRET) is a powerful tool to investigate biochemical and biophysical processes in
vitro and in vivo. We present the computational study of a novel FRET system, namely, a fuse protein that is composed of two far-red fluorescent proteins FusionRed and eqFP670 joined with a linker (FusionRed-Linker1-eqFP670). The latter contains a tetrapeptide DEVD motif which can be specifically cleaved by caspase-3. FusionRed acts as an energy
donor and eqFP670 as an energy acceptor in this pair. We carried out the comprehensive study of the factors that
influence FRET efficiency, including, in particular, distances between donor and acceptor, as well as relative orientations of transition dipole moments from donor to acceptor and the structure of linker between donor and acceptor.
We started from the primary structure of the proteins and also used available data on the similar β-barrels to construct the full-atom 3D structure of FusionRed-Linker1-eqFP670 sensor. We used a protein-protein docking procedure to construct the tetramer structure of the fuse protein. We compared this sensor with another FLIM/FRET-based sensor on caspase-3, TagRFP-23-KFP and explained the differences in their properties.
Visualization of single molecules and specific subsets of cells is widely used for studies of biological processes and particularly in immunological research. Recent technological advances have provided a qualitative change in biological visualization from studying of "snapshot" pictures to real-time continuous observation of cellular dynamics in vivo. Contemporary methods of in vivo imaging make it possible to localize specific cells within organs and tissues, to study their differentiation, migration, and cell-to-cell interactions, and to follow some intracellular events. Fluorescence intravital microscopy plays an especially important role in high resolution molecular imaging. The methods of intravital microscopy are quickly advancing thanks to improvements in molecular sensors, labeling strategies, and detection approaches. Novel techniques allow simultaneous detection of various probes with better resolution and depth of imaging. In this review, we describe current methods for in vivo imaging, with special accent on fluorescence approaches, and discuss their applications for medical and biological studies.
Activatable fluorescent molecular probes are predominantly nonfluorescent in their inactivated state due to intramolecular quenching, but increase fluorescence yield significantly after enzyme-mediated hydrolysis of peptides. Continuous wave in vivo detection of these protease-activatable fluorophores in the heart, however, is limited by the inability to differentiate between activated and nonactivated fractions of the probe and is frequently complicated by large background signal from probe accumulation in the liver. Using a cathepsin-activatable near-infrared probe (PGC-800), we demonstrate here that fluorescence lifetime (FL) significantly increases in infarcted murine myocardial tissue (0.67 ns) when compared with healthy myocardium (0.59 ns) after 24 h. Furthermore, we show that lifetime contrast can be used to distinguish in vivo cardiac fluorescence from background nonspecific liver signal. The results of this study show that lifetime contrast is a helpful addition to preclinical imaging of activatable fluorophores in the myocardium by reporting molecular activity in vivo due to changes in intramolecular quenching. This characterization of FL from activatable molecular probes will be helpful for advancing in vivo imaging of enzyme activity.
We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.
Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters. Imaging of fluorescence in vivo has become the most commonly used radiological tool in all pre-clinical work. In the last decade, full-body pre-clinical imaging systems have emerged with a wide range of utilities and niche application areas. The range of fluorescent probes that can be excited in the visible to near-infrared part of the electromagnetic spectrum continues to expand, with the most value for in vivo use being beyond the 630 nm wavelength, because the absorption of light sharply decreases. Whole-body in vivo fluorescence imaging has not yet reached a state of maturity that allows its routine use in the scope of large-scale pre-clinical studies. This is in part due to an incomplete understanding of what the actual fundamental capabilities and limitations of this imaging modality are. However, progress is continuously being made in research laboratories pushing the limits of the approach to consistently improve its performance in terms of spatial resolution, sensitivity and quantification. This paper reviews this imaging technology with a particular emphasis on its potential uses and limitations, the required instrumentation, and the possible imaging geometries and applications. A detailed account of the main commercially available systems is provided as well as some perspective relating to the future of the technology development. Although the vast majority of applications of in vivo small animal imaging are based on epi-illumination planar imaging, the future success of the method relies heavily on the design of novel imaging systems based on state-of-the-art optical technology used in conjunction with high spatial resolution structural modalities such as MRI, CT or ultrasound.
The lifetime of a fluorophore generally varies in different environments, making the molecule a sensitive indicator of tissue oxygenation, pH, and glucose. However, lifetime measurements are complicated when the fluorophore is embedded in an optically thick, highly scattering medium such as human tissue. We formulate the inverse problem for fluorescence lifetime tomography using diffuse photon density waves, and we demonstrate the technique by deriving spatial images of heterogeneous fluorophore distribution and lifetime, using simulated measurements in heterogeneous turbid media.
We have imaged, in real time, fluorescent tumors growing and metastasizing in live mice. The whole-body optical imaging system
is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact
animals. We have established new human and rodent tumors that stably express very high levels of the Aequorea victoria green fluorescent protein (GFP) and transplanted these to appropriate animals. B16F0-GFP mouse melanoma cells were injected
into the tail vein or portal vein of 6-week-old C57BL/6 and nude mice. Whole-body optical images showed metastatic lesions
in the brain, liver, and bone of B16F0-GFP that were used for real time, quantitative measurement of tumor growth in each
of these organs. The AC3488-GFP human colon cancer was surgically implanted orthotopically into nude mice. Whole-body optical
images showed, in real time, growth of the primary colon tumor and its metastatic lesions in the liver and skeleton. Imaging
was with either a trans-illuminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color
charge-coupled device camera. The depth to which metastasis and micrometastasis could be imaged depended on their size. A
60-μm diameter tumor was detectable at a depth of 0.5 mm whereas a 1,800-μm tumor could be visualized at 2.2-mm depth. The
simple, noninvasive, and highly selective imaging of growing tumors, made possible by strong GFP fluorescence, enables the
detailed imaging of tumor growth and metastasis formation. This should facilitate studies of modulators of cancer growth including
inhibition by potential chemotherapeutic agents.
It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.
Recent advances in fluorescence resonance energy transfer have led to qualitative and quantitative improvements in the technique, including increased spatial resolution, distance range, and sensitivity. These advances, due largely to new fluorescent dyes, but also to new optical methods and instrumentation, have opened up new biological applications.
Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.
Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.
GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Förster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.
The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biological imaging. With no unified standard for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use? Which are the brightest? What additional factors determine which are best for a given experiment? Although in many cases, a trial-and-error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
Förster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.
• biosensor
• fluorescence lifetime imaging microscopy
• ubiquitin
• proteasome
The development of highly efficient analytical methods capable of probing biological systems at system level is an important task that is required in order to meet the requirements of the emerging field of systems biology. Optical molecular imaging (OMI) is a very powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. In this article, recent advances in OMI are reviewed extensively, such as the development of molecular probes that make imaging brighter, more stable and more informative (e.g., FPs and semiconductor nanocrystals, also referred to as quantum dots), the development of imaging approaches that provide higher resolution and greater tissue penetration, and applications for measuring biological events from molecule to organism level, including gene expression, protein and subcellular compartment localization, protein activation and interaction, and low-mass molecule dynamics. These advances are of great significance in the field of biological science and could also be applied to disease diagnosis and pharmaceutical screening. Further developments in OMI for systems biology are also proposed.
Genetically-coded, fluorescence resonance energy transfer (FRET) biosensors are widely used to study molecular events from single cells to whole organisms. They are unique among biosensors because of their spontaneous fluorescence and targeting specificity to both organelles and tissues. In this review, we discuss the theoretical basis of FRET with a focus on key parameters responsible for designing FRET biosensors that have the highest sensitivity. Next, we discuss recent applications that are grouped into four common biosensor design patterns--intermolecular FRET, intramolecular FRET, FRET from substrate cleavage and FRET using multiple colour fluorescent proteins. Lastly, we discuss recent progress in creating fluorescent proteins suitable for FRET purposes. Together these advances in the development of FRET biosensors are beginning to unravel the interconnected and intricate signalling processes as they are occurring in living cells and organisms.
Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.
Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M 13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.
Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (+/-5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.
Förster resonance energy transfer-based analytical techniques represent a unique tool for bioanalysis because they allow one to detect protein–protein interactions and conformational changes of biomolecules at the nanometer scale, both “in vitro” and “in vivo” in cells, tissues and organisms. These techniques are applied in diverse fields, from the detection and quantification of ligands able to bind to proteins or receptors to the development of RET-based whole-cell biosensors, microscope imaging techniques and “in vivo” whole-body imaging for the monitoring of physiological and pathological processes. However, their quantitative performances need further improvements and, even though RET measurement principles and procedures have been continuously improved, in some cases only qualitative or semiquantitative information can be obtained. In this review we report recent applications of RET-based analytical techniques and discuss their advantages and limitations.
Figure
RET-based techniques allow analysis of protein–protein interactions and conformational changes of biomolecules at the nanometer scale
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.
Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.
Optical sensing of specific molecular targets and pathways deep inside living mice has become possible as a result of a number of advances. These include design of biocompatible near-infrared fluorochromes, development of targeted and activatable 'smart' imaging probes, engineered photoproteins and advances in photon migration theory and reconstruction. Together, these advances will provide new tools making it possible to understand more fully the functioning of protein networks, diagnose disease earlier and speed along drug discovery.
Biophotonics techniques, especially those involving fluorescence, are widely used in proteomics to characterize the in vitro interactions between proteins in high-throughput mode. On the other hand, fluorescence-based imaging studies often show that protein activity is regulated through large protein complexes that transiently form at specific sites in the cell. One could therefore argue that a systematic functional analysis of the human proteome requires technologies that are capable of time and spatially resolved, multiplexed analysis of protein interactions within cells.
When the nonfluorescent chromoprotein asFP595 from Anemonia sulcata is subjected to sufficiently intense illumination near the absorbance maximum (lambda(abs)(max) = 568 nm), it undergoes a remarkable transition, termed "kindling", to a long-lived fluorescent state (lambda(em)(max) = 595 nm). In the dark recovery phase, the kindled state relaxes thermally on a time scale of seconds or can instantly be reverted upon illumination at 450 nm. The kindling phenomenon is enhanced by the Ala143 --> Gly point mutation, which slows the dark recovery time constant to 100 s at room temperature and increases the fluorescence quantum yield. To investigate the chemical nature of the chromophore and the possible role of chromophore isomerization in the kindling phenomenon, we determined the crystal structure of the "kindling fluorescent protein" asFP595-A143G (KFP) in the dark-adapted state at 1.38 A resolution and 100 K. The chromophore, derived from the Met63-Tyr64-Gly65 tripeptide, closely resembles that of the nonfluorescent chromoprotein Rtms5 in that the configuration is trans about the methylene bridge and there is substantial distortion from planarity. Unlike in Rtms5, in the native protein the polypeptide backbone is cleaved between Cys62 and Met63. The size and shape of the chromophore pocket suggest that the cis isomer of the chromophore could also be accommodated. Within the pocket, partially disordered His197 displays two conformations, which may constitute a binary switch that stabilizes different chromophore configurations. The energy barrier for thermal relaxation was found by Arrhenius plot analysis to be approximately 71 kJ/mol, somewhat higher than the value of approximately 55 kJ/mol observed for cis-trans isomerization of a model chromophore in solution.
Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM systems have been implemented both in the frequency domain, using sinusoidally intensity-modulated excitation light and modulated detectors, and in the time domain, using pulsed excitation sources and time-correlated or time-gated detection. In this review we describe the different modes in which both frequency-domain and time-domain FLIM instruments have been constructed in wide-field and in point-scanning (confocal) microscopes. Also, novel additional strategies for constructing FLIM-instruments are discussed. In addition to technical implementation, this chapter gives an overview of the application of FLIM in cell biological en biomedical studies. Especially for in situ protein-protein interaction studies using fluorescence resonance energy transfer (FRET), FLIM has proven to be a robust and established technique in modern cell biology. Other application areas, including usage of lifetime contrast for ion-imaging, quantitative imaging, tissue characterization and medical applications, are discussed.
Naturally fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly. These proteins have allowed us to visualize, in real time, important aspects of cancer in living animals, including tumour cell mobility, invasion, metastasis and angiogenesis. These multicoloured proteins have allowed the colour-coding of cancer cells growing in vivo and enabled the distinction of host from tumour with single-cell resolution. Visualization of many aspects of cancer initiation and progression in vivo should be possible with fluorescent proteins.
Fluorescence lifetime imaging can provide valuable diagnostic information relating to the functional status of diseases. In this study, a near-infrared (NIR) dye-labeled hexapeptide (abbreviated Cyp-GRD) was synthesized. In vitro, Cyp-GRD internalized in nonsmall cell lung cancer cells (A549) without observable cytotoxic or proliferative effects to the cells at a concentration up to 1x10(-4) M. Time-domain fluorescence intensity and lifetime imaging of Cyp-GRD injected into A549 tumor-bearing mice revealed that the probe preferentially accumulated in the tumor and the major excretion organs. The fluorescence lifetime of the conjugate at the tumor site was mapped, showing the spatial distribution of the lifetime related to its environment. Additionally, fluorescence intensity image reconstruction obtained by integrating the time-resolved intensities enabled the contrast ratios of tumor-to-kidney or liver in slices at different depths to be displayed. The mean lifetime was 1.03 ns for the tumor and 0.80 ns for the liver when averaging those pixels exhibiting adequate signal-to-noise ratio, showing the tumor had a higher lifetime average and reflecting the altered physiopathology of the tumor. This study clearly demonstrated the feasibility of whole-body NIR fluorescence lifetime imaging for tumor localization and its spatial functional status in living small animals.
Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.
Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent
probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study
proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization,
activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic
fluorescent dyes, nanocrystals (“quantum dots”), autofluorescent proteins, small genetic encoded tags that can be complexed
with fluorochromes, and combinations of these probes are highlighted.
Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.
The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.
To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. The Olympus IV100 Laser Scanning Microscope, with ultra-narrow microscope objectives ("stick objectives"), is used for three-color whole-body imaging of the two-color cancer cells interacting with the GFP-expressing stromal cells. In this model, drug response of both cancer and stromal cells in the intact live animal is also imaged in real time. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow. This new model system enables the first cellular and subcellular images of unperturbed tumors in the live intact animal. New visible real-time targets for novel anticancer agents are provided in this model, including the color-coded interacting cancer and stromal cells, tumor vasculature, and blood flow. This imageable model should lead to many new insights of in vivo cancer cell biology and to novel drug discovery.
- E B Van Munster
- T W Gadella
E. B. van Munster and T. W. Gadella, Adv. Biochem.
Eng. Biotechnol. 95, 143-175 (2005).
- W Du
- Y Wang
- Q Luo
- B.-F Liu
W. Du, Y. Wang, Q. Luo, and B.-F. Liu, Anal. Bioanal. Biochem. 386, 444-457 (2006).
- A Roda
- M Guardigli
- E Michelini
- M Mirasoli
A. Roda, M. Guardigli, E. Michelini, and M. Mirasoli,
Anal. Bioanal. Chem. 393, 109-123 (2009).
- A G Harpur
- F S Wouters
- P I H Bastiaens
A. G. Harpur, F. S. Wouters, and P. I. H. Bastiaens,
Nature Biotechnol. 19, 167-169 (2001).
- K A Lukyanov
- A F Fradkov
- N G Gurskaya
- M V Matz
- Y A Labas
- A P Savitsky
- M L Markelov
- A G Zaraisky
- X Zhao
- Y Fang
- W Tan
- S A Lukyanov
K. A. Lukyanov, A. F. Fradkov, N. G. Gurskaya, M. V.
Matz, Y. A. Labas, A. P. Savitsky, M. L. Markelov, A. G.
Zaraisky, X. Zhao, Y. Fang, W. Tan, and S. A. Lukyanov, J. Biol. Chem. 275, 25879–25872 (2000).
- S Karasawa
- T Araki
- T Nagai
- H Mizuno
- A Miyawaki
S. Karasawa, T. Araki, T. Nagai, H. Mizuno, and
A. Miyawaki, Biochem. J. 381, 307-312 (2004).
- Y Yan
- G Marriott
Y. Yan and G. Marriott, J. Cur. Opin. Chem. Biol. 7,
635-640 (2003).
- V I Martynov
- A P Savitsky
- N Y Martynova
- P A Savitsky
- K A Lukyanov
- S A Lukyanov
V. I. Martynov, A. P. Savitsky, N. Y. Martynova, P. A.
Savitsky, K. A. Lukyanov, and S. A. Lukyanov, J.
Biol. Chem. 276, 21012–21016 (2001).
- I T Li
- E Pham
- K Truong
I. T. Li, E. Pham, and K. Truong, J. Biotechnol. Lett.
28, 1971-1982 (2006).
- D M Chudakov
- V V Belousov
- A G Zaraisky
- V V Novoselov
- D B Staroverov
- D B Zorov
- S Lukyanov
- K A Lukyanov
D. M. Chudakov, V. V. Belousov, A. G. Zaraisky, V. V.
Novoselov, D. B. Staroverov, D. B. Zorov, S. Lukyanov, and K. A. Lukyanov, Nature Biotechnol. 21,
191-194 (2003).
- R Y Tsien
R. Y. Tsien, Nature Rev. Molec. Cell Biol. 4, SS16-SS21 (2003).
- N C Shaner
- P A Steinbach
- R Y Tsien
N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat.
Methods 2, 905-909 (2007).
- K Truong
- M Ikura
K. Truong and M. Ikura, Cur. Opin. Struct. Biol. 11,
573-578 (2001).
- M Hassan
- J Riley
- V Chernomordik
- P Smith
- R Pursley
- S B Lee
- J Capala
- A H Gandjbakhche
M. Hassan, J. Riley, V. Chernomordik, P. Smith,
R. Pursley, S. B. Lee, J. Capala, and A. H. Gandjbakhche, Mol. Imag. 6, 229-236 (2007).
- B N G Giepmans
- S R Adams
- M H Ellisman
- R Y Tsien
B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and
R. Y. Tsien, Science 312, 217-224 (2006).
- R Weissleder
- V Ntziachristos
- Shedding
R. Weissleder and V. Ntziachristos, Shedding, Nature
Med. 9, 123-128 (2003).
- P R Selvin
P. R. Selvin, Nature Struct. Biol. 7, 730-734 (2000).
Molecular Cloning: a Laboratory Manual
- J Sambrook
- E F Fritisch
- T Maniatis
J. Sambrook, E. F. Fritisch, and T. Maniatis, Molecular Cloning: a Laboratory Manual, 2nd edn. (Cold
Spring Harbor Laboratory Press, New York, 1989).