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Vladislav V. Verkhusha - One of the best experts on this subject based on the ideXlab platform.
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Near-Infrared Fluorescent Proteins and Their Applications
Biochemistry (Moscow), 2019Co-Authors: M. M. Karasev, O. V. Stepanenko, K. A. Rumyantsev, K. K. Turoverov, Vladislav V. VerkhushaAbstract:High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650–900 nm) open a possibility for in vivo imaging of biological processes at the micro-and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes–natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these Proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded Fluorescent probes, similarly to GFP-like Fluorescent Proteins. In this review, we discuss photophysical and biochemical properties of NIR Fluorescent Proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR Fluorescent probes are discussed. Applications of NIR Fluorescent Proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo , are described. We specifically focus on the use of NIR Fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.
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a palette of Fluorescent Proteins optimized for diverse cellular environments
Nature Communications, 2015Co-Authors: Lindsey M Costantini, Vladislav V. Verkhusha, Mikhail Baloban, Michele L Markwardt, Mark A Rizzo, Erik L SnappAbstract:Quantitative live cell imaging of protein trafficking suffers from misfolding and inappropriate disulphide bond formation of Fluorescent Proteins in the secretory pathway. Here, the authors present an optimized collection of Fluorescent Proteins suitable for use in oxidizing subcellular compartments.
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photocontrollable Fluorescent Proteins for superresolution imaging
Annual Review of Biophysics, 2014Co-Authors: Daria M Shcherbakova, Prabuddha Sengupta, Jennifer Lippincottschwartz, Vladislav V. VerkhushaAbstract:Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable Fluorescent Proteins. The fluorescence of these Proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable Fluorescent Proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable Fluorescent Proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable Proteins in superresolution imaging, as well as how these ap...
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Multicontrast photoacoustic in vivo imaging using near-infrared Fluorescent Proteins
Scientific Reports, 2014Co-Authors: Arie Krumholz, Daria M Shcherbakova, Lihong V. Wang, Vladislav V. VerkhushaAbstract:Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared Fluorescent Proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted Fluorescent Proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo .
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near infrared Fluorescent Proteins for multicolor in vivo imaging
Nature Methods, 2013Co-Authors: Daria M Shcherbakova, Vladislav V. VerkhushaAbstract:Four spectrally distinct near-infrared Fluorescent Proteins based on bacterial phytochromes are described, expanding the possibilities for multicolor in vivo imaging experiments in nontransparent organisms.
Roger Y Tsien - One of the best experts on this subject based on the ideXlab platform.
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Improving FRET dynamic range with bright green and red Fluorescent Proteins
Nature Methods, 2012Co-Authors: François St-pierre, Michelle A. Baird, Michael W Davidson, Michael R. Mckeown, Yiyang Gong, Jesse D Marshall, Paula J Cranfill, Jörg Wiedenmann, Mark J Schnitzer, Roger Y TsienAbstract:Development of the bright green and red Fluorescent Proteins, Clover and mRuby2, creates a fluorescence resonance energy transfer (FRET) pair with the highest Förster radius among existing ratiometric FRET pairs. Substitution of this pair for current FRET pairs in several existing sensors reliably and substantially improves sensor performance. A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two Fluorescent Proteins, Clover and mRuby2, which are the brightest green and red Fluorescent Proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two Proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.
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mammalian expression of infrared Fluorescent Proteins engineered from a bacterial phytochrome
Science, 2009Co-Authors: Antoine Royant, Paul Steinbach, Todd A Aguilera, Varda Levram, Roger Y TsienAbstract:Visibly Fluorescent Proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans , incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-Fluorescent Proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M −1 cm −1 ; and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.
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Evaluating and improving the photostability of Fluorescent Proteins
Fluorescence In Vivo Imaging Based on Genetically Engineered Probes: From Living Cells to Whole Body Imaging IV, 2009Co-Authors: Nathan C. Shaner, Kristin L Hazelwood, Michael W Davidson, Michael R. Mckeown, Paul Steinbach, Roger Y TsienAbstract:Fluorescent Proteins are the most common and versatile class of genetically encoded optical probes. While structure-guided rational design and directed evolution approaches have largely overcome early problems such as oligomerization, poor folding at physiological temperatures, and availability of wavelengths suitable for multi-color imaging, nearly all Fluorescent Proteins have yet to be fully optimized. We have developed novel methods for evaluating the current generation of Fluorescent Proteins and improving their remaining suboptimal properties. Little is yet known about the mechanisms responsible for photobleaching of Fluorescent Proteins, and inadequate photostability is a chief complaint among end users. In order to compare the performance of Fluorescent Proteins across the visual spectrum, we have standardized a method used to measure photostability in live cells under both widefield and confocal laser illumination. This method has allowed us to evaluate a large number of commonly used Fluorescent Proteins, and has uncovered surprisingly complex and unpredictable behaviors in many of these Proteins. We have also developed novel methods for selecting explicitly for high photostability during the directed evolution process, leading to the development of highly improved monomeric orange and red Fluorescent Proteins. These Proteins, most notably our photostable derivative of TagRFP, have remarkably high photostability and have proven useful as fusion tags for long-term imaging. Our methods should be applicable to any of the large number of Fluorescent Proteins still in need of improved photostability.
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improving the photostability of bright monomeric orange and red Fluorescent Proteins
Nature Methods, 2008Co-Authors: Nathan C. Shaner, Kristin L Hazelwood, Michael W Davidson, Michael R. Mckeown, Paul Steinbach, Roger Y TsienAbstract:Improved photostability of Fluorescent Proteins would benefit many applications but is usually an afterthought in selection screens. Setting photostability as the primary selection criterion in screens for improved Fluorescent Proteins yielded highly photostable variants of existing orange and red Fluorescent Proteins without compromising other beneficial characteristics. All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although Fluorescent Proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting Fluorescent Proteins. Here we describe an assay for screening libraries of Fluorescent Proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original Proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.
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A guide to choosing Fluorescent Proteins
Nature Methods, 2005Co-Authors: Nathan C. Shaner, Paul A. Steinbach, Roger Y TsienAbstract: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.
Robert M. Hoffman - One of the best experts on this subject based on the ideXlab platform.
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4.03 – Fluorescent Proteins
Comprehensive Biomedical Physics, 2020Co-Authors: Robert M. HoffmanAbstract:The tumor microenvironment (TME) is comprised of interacting cancer and stromal cells. The TME plays a major role in tumor behavior. This chapter reviews the development of three different colors of transgenic mice ubiquitously expressing either red Fluorescent protein, green Fluorescent protein, or cyan Fluorescent protein for use to color the tumor stroma. This chapter demonstrates how cancer cells, expressing various colors of Fluorescent Proteins, implanted in the colored nude mice, enable the real-time imaging of the cancer cell–stromal cell interaction in real time in live mice. This technology described here demonstrated that stromal cells are necessary for cancer cells to metastasize. The TME consists of both cancer cells and stromal cells. Their interaction has major effects on tumor behavior, including the ability to metastasize. To understand cancer cell–stromal cell interaction, it is necessary to be able to differentiate by imaging these cell types which is readily done with the use of Fluorescent Proteins. This is the theme of the present chapter on a multicolor imaging model of cancer cell/stromal cell interaction.
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Fluorescent Proteins as Sensors for Cellular Behavior in Mice.
Progress in Molecular Biology and Translational Science, 2018Co-Authors: Robert M. HoffmanAbstract:Abstract Imaging of cancer cells in mice expressing Fluorescent Proteins has allowed the real-time tracing of cancer growth and metastasis and determination of efficacy of candidate antitumor and antimetastatic agents, especially in mouse orthotopic models. The use of Fluorescent Proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear–cytoplasmic dynamics of cancer cells in vivo, including mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer cell deformation, migration, and extravasation. Recent applications of the technology described here include linking Fluorescent Proteins with cell cycle-specific Proteins such that the cells change color from red to green as they transit from G1 to S phases. Any in vivo process can be imaged using Fluorescent Proteins, allowing molecular biology to advance from in vitro studies to studying molecular processes in the living animal.
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Strategies for In Vivo Imaging Using Fluorescent Proteins.
Journal of Cellular Biochemistry, 2017Co-Authors: Robert M. HoffmanAbstract:: Fluorescent Proteins have enabled the color-coding of cells growing in vivo. Noninvasive imaging of cells expressing Fluorescent Proteins has allowed the real-time determination of the behavior of cancer cells, the progression of infection, the differentiation of stem cells, and interaction of stromal and cancer cells. Cancer cells labeled in the nucleus and cytoplasm with spectrally-distinct Proteins can visualize in vivo nuclear-cytoplasmic dynamics in vivo including: mitosis, apoptosis, cell-cycle phase, and differential behavior of nucleus and cytoplasm that occurs during cancer-cell deformation. Linking spectrally-distinct Fluorescent Proteins with cell-cycle-specific Proteins results in color-coding the phases of the cell cycle. With the use of Fluorescent Proteins, literally any cellular or molecular function can be imaged in vivo. J. Cell. Biochem. 118: 2571-2580, 2017. © 2017 Wiley Periodicals, Inc.
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Fluorescent Proteins as visible in vivo sensors.
Progress in Molecular Biology and Translational Science, 2012Co-Authors: Robert M. HoffmanAbstract:Abstract Fluorescent Proteins have enabled a whole new technology of visible in vivo genetic sensors. 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 tumor cell mobility, invasion, metastasis, and angiogenesis. These multicolored Proteins have allowed the color coding of cancer cells growing in vivo and enabled the distinction of host from tumor with single-cell resolution. Whole-body imaging with Fluorescent Proteins has been shown to be a powerful technology to noninvasively follow the dynamics of metastatic cancer. Whole-body imaging of cancer cells expressing Fluorescent Proteins has enabled the facile determination of efficacy of candidate antitumor and antimetastatic agents in mouse models. The use of Fluorescent Proteins to differentially label cancer cells in the nucleus and cytoplasm and high-powered imaging technology have enabled the visualization of the nuclear–cytoplasmic dynamics of cancer cells in vivo, including noninvasive techniques. Fluorescent Proteins thus enable both macro- and microimaging technology and thereby provide the basis for the new field of in vivo cell biology.
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Cellular and subcellular imaging in live mice using Fluorescent Proteins.
Current Pharmaceutical Biotechnology, 2012Co-Authors: Robert M. HoffmanAbstract:: Fluorescent Proteins have revolutionized in vivo biology. Due to their intrinsic brightness, multiple colors, and ease of genetic manipulation, Fluorescent Proteins have been demonstrated to be the reporters of choice for in vivo imaging. The present report reviews applications of Fluorescent Proteins for imaging cancer progression, gene expression, angiogenesis, stem cells, bacterial infection, Leishmania infection, and asthma, at the cellular and subcellular level in live mice. With Fluorescent-protein-expressing cells and a highly sensitive small animal imaging system, cellular and subcellular dynamics can now be observed in live mice in real time. Such imaging possibilities can provide new visual targets for novel drug therapy. Fluorescent Proteins thus enable both micro as well as macro imaging technology and thereby provide the basis for the new field of in vivo cell biology.
Vasilis Ntziachristos - One of the best experts on this subject based on the ideXlab platform.
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in vivo tomographic imaging of red shifted Fluorescent Proteins
Biomedical Optics Express, 2011Co-Authors: Nikolaos C Deliolanis, Thomas Wurdinger, Lisa Pike, Bakhos A Tannous, Xandra O Breakefield, Ralph Weissleder, Vasilis NtziachristosAbstract:We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared Fluorescent Proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted Fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green Fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted Fluorescent Proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.
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multispectral opto acoustic tomography of deep seated Fluorescent Proteins in vivo
Nature Photonics, 2009Co-Authors: Daniel Razansky, Martin Distel, Claudio Vinegoni, Norbert Perrimon, Reinhard W Koster, Vasilis NtziachristosAbstract:Fluorescent Proteins have become essential reporter molecules for studying life at the cellular and sub-cellular level, re-defining the ways in which we investigate biology. However, because of intense light scattering, most organisms and tissues remain inaccessible to current fluorescence microscopy techniques at depths beyond several hundred micrometres. We describe a multispectral opto-acoustic tomography technique capable of high-resolution visualization of Fluorescent Proteins deep within highly light-scattering living organisms. The method uses multiwavelength illumination over multiple projections combined with selective-plane opto-acoustic detection for artifact-free data collection. Accurate image reconstruction is enabled by making use of wavelength-dependent light propagation models in tissue. By performing whole-body imaging of two biologically important and optically diffuse model organisms, Drosophila melanogaster pupae and adult zebrafish, we demonstrate the facility to resolve tissue-specific expression of eGFP and mCherrry Fluorescent Proteins for precise morphological and functional observations in vivo.
Gert Jan Kremers - One of the best experts on this subject based on the ideXlab platform.
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quantitative assessment of Fluorescent Proteins
Nature Methods, 2016Co-Authors: Michelle A. Baird, John R Allen, Paula J Cranfill, Brittney R Sell, Zeno Lavagnino, Martijn H De Gruiter, Gert Jan KremersAbstract:The advent of Fluorescent Proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autoFluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
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photoconversion in orange and red Fluorescent Proteins
Nature Methods, 2009Co-Authors: Gert Jan Kremers, Kristin L Hazelwood, Christopher S Murphy, Michael W Davidson, David W. PistonAbstract:Several red and orange Fluorescent Proteins are reported to be photoconvertible. Specifically, three red Fluorescent Proteins that can be switched to green, and two orange Fluorescent Proteins that can be switched to far red are reported.
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Turning Fluorescent Proteins into energy-saving light bulbs.
Nature methods, 2008Co-Authors: Gert Jan Kremers, David W. PistonAbstract:Screening for photostability in addition to color and brightness creates\nbetter Fluorescent Proteins.
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cyan and yellow super Fluorescent Proteins with improved brightness protein folding and fret forster radius
Biochemistry, 2006Co-Authors: Gert Jan Kremers, Joachim Goedhart, Erik B Van Munster, Theodorus W J GadellaAbstract: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 °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...