High-throughput microscopic screening instruments can generate huge collections of images of live cells incubated with combinatorial libraries of fluorescent molecules. Organizing and visualizing these images to discern biologically important patterns that link back to chemical structure is a challenge. We present an analysis and visualization methodology - Cheminformatic Assisted Image Array (CAIA) - that greatly facilitates data mining efforts. For illustration, we considered a collection of microscopic images acquired from cells incubated with each member of a combinatorial library of styryl molecules being screened for candidate bioimaging probes. By sorting CAIAs based on quantitative image features, the relative contribution of each combinatorial building block on probe intracellular distribution could be visually discerned. The results revealed trends hidden in the dataset: most interestingly, the building blocks of the styryl molecules appeared to behave as chemical address tags, additively and independently encoding spatial patterns of intracellular fluorescence. Translated into practice, CAIA facilitated discovery of several outstanding styryl molecules for live cell nuclear imaging applications.
Cheminformatics; high content screening; combinatorial library; styryl; fluorescence; bioimaging; chemical address tags; QSAR; CAIA
With a combinatorial library of bioimaging probes, it is now possible to use machine vision to analyze the contribution of different building blocks of the molecules to their cell-associated visual signals. For athis purpose, cell-permeant, fluorescent styryl molecules were synthesized by condensation of 168 aldehyde with 8 pyridinium/quinolinium building blocks. Images of cells incubated with fluorescent molecules were acquired with a high content screening instrument. Chemical and image feature analysis revealed how variation in one or the other building block of the styryl molecules led to variations in the molecules' visual signals. Across each pair of probes in the library, chemical similarity was significantly associated with spectral and total signal intensity similarity. However, chemical similarity was much less associated with similarity in subcellular probe fluorescence patterns. Quantitative analysis and visual inspection of pairs of images acquired from pairs of styryl isomers confirm that many closely-related probes exhibit different subcellular localization patterns. Therefore, idiosyncratic interactions between styryl molecules and specific cellular components greatly contribute to the subcellular distribution of the styryl probes' fluorescence signal. These results demonstrate how machine vision and cheminformatics can be combined to analyze the targeting properties of bioimaging probes, using large image data sets acquired with automated screening systems.
Cheminformatics; machine vision; bioimaging; fluorescence; styryl; high content screening; image cytometry; combinatorial chemistry
In recent years, new microscopic imaging techniques have evolved to allow us to visualize several different proteins (or other biomolecules) in a visual field. Analysis of protein co-localization becomes viable because molecules can interact only when they are located close to each other. We present a novel approach to align images in a multi-tag fluorescence image stack. The proposed approach is applicable to multi-tag bioimaging systems which (a) acquire fluorescence images by sequential staining and (b) simultaneously capture a phase contrast image corresponding to each of the fluorescence images. To the best of our knowledge, there is no existing method in the literature, which addresses simultaneous registration of multi-tag bioimages and selection of the reference image in order to maximize the overall overlap between the images.
We employ a block-based method for registration, which yields a confidence measure to indicate the accuracy of our registration results. We derive a shift metric in order to select the Reference Image with Maximal Overlap (RIMO), in turn minimizing the total amount of non-overlapping signal for a given number of tags. Experimental results show that the Robust Alignment of Multi-Tag Bioimages (RAMTaB) framework is robust to variations in contrast and illumination, yields sub-pixel accuracy, and successfully selects the reference image resulting in maximum overlap. The registration results are also shown to significantly improve any follow-up protein co-localization studies.
For the discovery of protein complexes and of functional protein networks within a cell, alignment of the tag images in a multi-tag fluorescence image stack is a key pre-processing step. The proposed framework is shown to produce accurate alignment results on both real and synthetic data. Our future work will use the aligned multi-channel fluorescence image data for normal and diseased tissue specimens to analyze molecular co-expression patterns and functional protein networks.
To study the chemical determinants of small molecule transport inside cells, it is crucial to visualize relationships between the chemical structure of small molecules and their associated subcellular distribution patterns. For this purpose, we experimented with cells incubated with a synthetic combinatorial library of fluorescent, membrane-permeant small molecule chemical agents. With an automated high content screening instrument, the intracellular distribution patterns of these chemical agents were microscopically captured in image data sets, and analyzed off-line with machine vision and cheminformatics algorithms. Nevertheless, it remained challenging to interpret correlations linking the structure and properties of chemical agents to their subcellular localization patterns in large numbers of cells, captured across large number of images.
To address this challenge, we constructed a Multidimensional Online Virtual Image Display (MOVID) visualization platform using off-the-shelf hardware and software components. For analysis, the image data set acquired from cells incubated with a combinatorial library of fluorescent molecular probes was sorted based on quantitative relationships between the chemical structures, physicochemical properties or predicted subcellular distribution patterns. MOVID enabled visual inspection of the sorted, multidimensional image arrays: Using a multipanel desktop liquid crystal display (LCD) and an avatar as a graphical user interface, the resolution of the images was automatically adjusted to the avatar’s distance, allowing the viewer to rapidly navigate through high resolution image arrays, zooming in and out of the images to inspect and annotate individual cells exhibiting interesting staining patterns. In this manner, MOVID facilitated visualization and interpretation of quantitative structure-localization relationship studies. MOVID also facilitated direct, intuitive exploration of the relationship between the chemical structures of the probes and their microscopic, subcellular staining patterns.
MOVID can provide a practical, graphical user interface and computer-assisted image data visualization platform to facilitate bioimage data mining and cheminformatics analysis of high content, phenotypic screening experiments.
Machine vision; Cheminformatics; Virtual reality; Data mining; Optical probes; Multivariate analysis; Human-computer interaction; Graphical user interface
Motivation: Bioimaging techniques rapidly develop toward higher resolution and dimension. The increase in dimension is achieved by different techniques such as multitag fluorescence imaging, Matrix Assisted Laser Desorption / Ionization (MALDI) imaging or Raman imaging, which record for each pixel an N-dimensional intensity array, representing local abundances of molecules, residues or interaction patterns. The analysis of such multivariate bioimages (MBIs) calls for new approaches to support users in the analysis of both feature domains: space (i.e. sample morphology) and molecular colocation or interaction. In this article, we present our approach WHIDE (Web-based Hyperbolic Image Data Explorer) that combines principles from computational learning, dimension reduction and visualization in a free web application.
Results: We applied WHIDE to a set of MBI recorded using the multitag fluorescence imaging Toponome Imaging System. The MBI show field of view in tissue sections from a colon cancer study and we compare tissue from normal/healthy colon with tissue classified as tumor. Our results show, that WHIDE efficiently reduces the complexity of the data by mapping each of the pixels to a cluster, referred to as Molecular Co-Expression Phenotypes and provides a structural basis for a sophisticated multimodal visualization, which combines topology preserving pseudocoloring with information visualization. The wide range of WHIDE's applicability is demonstrated with examples from toponome imaging, high content screens and MALDI imaging (shown in the Supplementary Material).
Availability and implementation: The WHIDE tool can be accessed via the BioIMAX website http://ani.cebitec.uni-bielefeld.de/BioIMAX/; Login: whidetestuser; Password: whidetest.
Supplementary data are available at Bioinformatics online.
A two-photon absorbing (2PA) and aggregation-enhanced near infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong signal as the unaggregated dye, a three-fold increase in two-photon absorption relative to the DFP in solution, and approx. four-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and 1H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FRtargeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 µm deep in the HeLa tumor.
Aggregation enhanced emission; near infrared emission; folate receptor targeting; two-photon absorption; two-photon fluorescence microscopy; silica nanoparticles; HeLa tumor; in vivo imaging; ex vivo imaging
In recent years, the deluge of complicated molecular and cellular microscopic images creates compelling challenges for the image computing community. There has been an increasing focus on developing novel image processing, data mining, database and visualization techniques to extract, compare, search and manage the biological knowledge in these data-intensive problems. This emerging new area of bioinformatics can be called ‘bioimage informatics’. This article reviews the advances of this field from several aspects, including applications, key techniques, available tools and resources. Application examples such as high-throughput/high-content phenotyping and atlas building for model organisms demonstrate the importance of bioimage informatics. The essential techniques to the success of these applications, such as bioimage feature identification, segmentation and tracking, registration, annotation, mining, image data management and visualization, are further summarized, along with a brief overview of the available bioimage databases, analysis tools and other resources.
Supplementary information: Supplementary data are available at Bioinformatics online.
Fluorescent probes, which allow visualization of cations such as Ca2+, Zn2+ etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.
probe; bioimaging; photoinduced electron transfer; fluorescence; spirocyclization
Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.
We have developed an integrative, cell-based modeling approach to facilitate the design and discovery of chemical agents directed to specific sites of action within a living organism. Here, a computational, multiscale transport model of the lung was adapted to enable virtual screening of small molecules targeting the epithelial cells of the upper airways. In turn, the transport behaviors of selected candidate probes were evaluated to establish their degree of retention at a site of absorption, using computational simulations as well as two in vitro cell-based assay systems. Lastly, bioimaging experiments were performed to examine candidate molecules' distribution in the lungs of mice after local and systemic administration. Based on computational simulations, the higher mitochondrial density per unit absorption surface area is the key parameter determining the higher retention of small molecule hydrophilic cations in the upper airways, relative to lipophilic weak bases, specifically after intratracheal administration.
The steady-state photophysical, NMR, and two-photon absorption (2PA) properties of a new fluorene derivative (1) containing the 2-(2′-hydroxyphenyl) benzothiazole (HBT) terminal construct was investigated for use as a fluorescence probe in bioimaging. A comprehensive analysis of the linear spectral properties revealed inter- and intramolecular hydrogen bonding and excited state intramolecular proton transfer (ESIPT) processes in the HBT substituent. A specific electronic model with a double minimum potential energy surface was consistent with the observed spectral properties. The 2PA spectra were obtained using a standard two-photon induced fluorescence method with femtosecond kHz laser system, affording a maximum 2PA cross section of ~600 GM, a sufficiently high value for two-photon fluorescence imaging. No dependence of two-photon absorption efficiency on solvent properties and hydrogen bonding in the HBT substituent was observed. The potential use of this fluorenyl probe in bioimaging was demonstrated via one- and two-photon fluorescence imaging of COS-7 cells.
Fluorescence probes; imaging agents; hydrogen bonding; two-photon induced fluorescence
This paper reviews image processing and pattern recognition techniques, which will be useful to analyze bioimages. Although this paper does not provide their technical details, it will be possible to grasp their main tasks and typical tools to handle the tasks. Image processing is a large research area to improve the visibility of an input image and acquire some valuable information from it. As the main tasks of image processing, this paper introduces gray-level transformation, binarization, image filtering, image segmentation, visual object tracking, optical flow and image registration. Image pattern recognition is the technique to classify an input image into one of the predefined classes and also has a large research area. This paper overviews its two main modules, that is, feature extraction module and classification module. Throughout the paper, it will be emphasized that bioimage is a very difficult target for even state-of-the-art image processing and pattern recognition techniques due to noises, deformations, etc. This paper is expected to be one tutorial guide to bridge biology and image processing researchers for their further collaboration to tackle such a difficult target.
bioimage informatics; image pattern recognition; image processing
Over the last twenty years there have been great advances in light microscopy with the result that multi-dimensional imaging has driven a revolution in modern biology. The development of new approaches of data acquisition are reportedly frequently, and yet the significant data management and analysis challenges presented by these new complex datasets remains largely unsolved. Like the well-developed field of genome bioinformatics, central repositories are and will be key resources, but there is a critical need for informatics tools in individual laboratories to help manage, share, visualize, and analyze image data. In this article we present the recent efforts by the bioimage informatics community to tackle these challenges and discuss our own vision for future development of bioimage informatics solution.
microscopy; file formats; image management; image analysis; image processing
The synthesis, linear photophysical and photochemical parameters, two-photon absorption (2PA), and superfluorescence properties of 2,2′-(5,5′-(9,9-didecyl-9H-fluorene-2,7-diyl)bis(ethyne-2,1-diyl)bis(thiophene-5,2-diyl))dibenzo[d]thiazole (1) were investigated, suggesting its potential as an efficient fluorescent probe for bioimaging applications. The steady-state absorption, fluorescence, and excitation anisotropy spectra of 1 were measured in several organic solvents and aqueous media. Probe 1 exhibited high fluorescence quantum yield (∼ 0.7-0.8) and photochemical stability (photobleaching quantum yield ∼ (3 - 7)·10-6). The 2PA spectra were determined over a broad spectral range (640-920 nm) using a standard two-photon induced fluorescence method under femtosecond excitation. A well-defined two-photon allowed absorption band at 680-720 nm with corresponding 2PA cross sections δ2PA ≈ 800-900 GM was observed. The use of probe 1 in bioimaging was shown via one- and two-photon fluorescence imaging of HCT-116 cells. An amplification of the stimulated emission of 1 was demonstrated in organic solvents and thin polystyrene films, which potentially can be used for the development of new fluorescent labels with increased spectral brightness.
Targeted molecular imaging with two-photon fluorescence microscopy (2PFM) is a powerful technique for chemical biology and, potentially, for non-invasive diagnosis and treatment of a number of diseases. The synthesis, photophysical studies, and bioimaging are reported for a versatile norbornene-based block copolymer multifunctional scaffold containing biocompatible (PEG), two-photon fluorescent dyes (fluorenyl), and targeting (cyclic-RGD peptide) moieties. The two bioconjugates, containing two different fluorenyl dyes and cRGDfK covalently attached to the polymer probe, formed a spherical micelle and self-assembled structure in water, for which size was analyzed by TEM and DLS. Cell-viability and 2PFM imaging of human epithelial U87MG cell lines that over express αvβ3 integrin was performed via incubation with the new probes, along with negative control studies using MCF-7 breast cancer cells and blocking experiments. 2PFM microscopy confirmed the high selectivity of the biocompatible probe in the integrin rich area in the U87MF cells while blocking as well as negative control MCF-7 experiments confirmed the integrin targeting ability of the new probes.
Water-soluble block copolymer probe; ROMP; two-photon bioimaging; integrin targeting
Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell.
Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs.
A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.
We present a biosegmentation benchmark that includes infrastructure, datasets with associated ground truth, and validation methods for biological image analysis. The primary motivation for creating this resource comes from the fact that it is very difficult, if not impossible, for an end-user to choose from a wide range of segmentation methods available in the literature for a particular bioimaging problem. No single algorithm is likely to be equally effective on diverse set of images and each method has its own strengths and limitations. We hope that our benchmark resource would be of considerable help to both the bioimaging researchers looking for novel image processing methods and image processing researchers exploring application of their methods to biology.
Our benchmark consists of different classes of images and ground truth data, ranging in scale from subcellular, cellular to tissue level, each of which pose their own set of challenges to image analysis. The associated ground truth data can be used to evaluate the effectiveness of different methods, to improve methods and to compare results. Standard evaluation methods and some analysis tools are integrated into a database framework that is available online at .
This online benchmark will facilitate integration and comparison of image analysis methods for bioimages. While the primary focus is on biological images, we believe that the dataset and infrastructure will be of interest to researchers and developers working with biological image analysis, image segmentation and object tracking in general.
Multimodal nonlinear optical (NLO) imaging is poised to become a powerful tool in bioimaging given its ability to capitalize on the unique advantages possessed by different NLO imaging modalities. The integration of different imaging modalities such as two-photon-excited fluorescence, sum frequency generation, and coherent anti-Stokes Raman scattering on the same platform can facilitate simultaneous imaging of different biological structures. Parameters to be considered in constructing a multimodal NLO microscope are discussed with emphasis on achieving a compromise in these parameters for efficient signal generation with each imaging modality. As an example of biomedical applications, multimodal NLO imaging is utilized to investigate the central nervous system in healthy and diseased states.
Central nervous system imaging; multimodality; nonlinear optical (NLO) microscopy
Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pre-tagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from.
In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole–alkene cycloaddition reaction (“photoclick chemistry”) and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M−1 s−1), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short “bridge” between two flanking phenyl rings.
This photoclick chemistry has been used to label proteins rapidly (within ~1 minute) both in vitro and in E. coli. To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.
Closely involved in the progression of nonlinear bioimaging is the development of optical probes for investigating biological function and activity. Introduction of new fluorescent compounds possessing enhanced nonlinearities are essential for advancing the utility of two-photon absorption (2PA) processes in the biological sciences. Herein, we report the synthesis of fluorene-based fluorophores tailored for multiphoton imaging, incorporating the succinimidyl ester and thioester functionality as reactive linkers for further coupling with a wide variety of biologically relevant molecules. The succinimidyl ester amine reactive probe was conjugated with the cyclic peptide RGDfK and polyclonal anti-rat IgG IgG protein. Upon conjugation, the basic molecular architecture and photophysical properties of the active 2PA chromophore remain unchanged. Conventional and two-photon fluorescence microscopy (2PFM) imaging of COS-7 and HeLa cells, incubated with either the fluorene-RGD peptide conjugate or the fluorene-IgG conjugate, was demonstrated. The fluorene-IgG conjugate was used to image cell spindles at early mitotic developmental stages.
Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.
Nanoparticle; Multiplex; Multiparameter; Plasmonics; Spectroscopy; Probe
Chemical tags for live cell imaging are emerging as viable alternatives to the fluorescent proteins for labeling proteins with small molecule probes. Among reported chemical tags, trimethoprim (TMP)-tag stands out for having sufficient cell permeability and selectivity to allow imaging of intracellular proteins. TMP-tag provides a non-covalent label in which the protein of interest is fused to E. coli dihydrofolate reductase (DHFR) and then labeled with a cell permeable TMP-probe heterodimer. To complement the utility of the non-covalent TMP-tag, we sought to render the TMP-tag covalent for applications such as single-molecule tracking and pulse-chase labeling that would benefit from a more permanent modification. Based on the long-standing use of proximity-induced reactivity for irreversible inhibitor design and its more recent application to in vitro chemical biology tools, we designed an eDHFR variant with a unique Cys residue positioned to react with an acrylamide electrophile installed on the TMP-probe label. In vitro experiments show that the eDHFR:Leu28Cys nucleophile reacts rapidly and quantitatively with the TMP-acrylamide-probe. Most significantly, the balance in reactivity provided by the acrylamide electrophile allows intracellular proteins tagged with eDHFR:Leu28Cys to be labeled with a TMP-acrylamide-fluorescein heterotrimer in live cells with minimal background. Thus, the TMP electrophile described here can be used immediately as a covalent chemical tag in live cells. Moreover, proximity-induced reactivity is shown to be sufficiently selective for use in a living cell, suggesting a general approach for the development of orthogonal covalent chemical tags from existing non-covalent ligand-protein pairs.
A variety of polymeric nanoparticles have been developed for bioimaging applications. In this study, we report use of a 50 nm recombinant protein nanoparticle with a multivalent surface as a vehicle for functionalization with a model imaging agent. Multiple fluorescent probes were covalently conjugated to surface amines of crosslinked amphiphilic elastin-mimetic protein micelles using N- hydroxysuccinimide ester chemistry. In vivo fluorescence imaging confirmed that protein micelles selectively accumulated at sites of angioplasty induced vessel wall injury presumably via an enhanced permeability and retention (EPR) effect. This investigation demonstrates the potential of amphiphilic protein micelles to be used as a vehicle for selective imaging of sites associated with disrupted or leaky endothelium.
polypeptides; protein micelles; conjugation; vascular permeability; bioimaging
Proteases play important roles in a variety of pathologies from heart disease to cancer. Quantitative measurement of protease activity is possible using a novel spectrally matched dual fluorophore probe and a small animal lifetime imager. The recorded fluorescence from an activatable fluorophore, one that changes its fluorescent amplitude after biological target interaction, is also influenced by other factors including imaging probe delivery and optical tissue absorption of excitation and emission light.
Fluorescence from a second spectrally matched constant (non-activatable) fluorophore on each nanoparticle platform can be used to correct for both probe delivery and tissue absorption. The fluorescence from each fluorophore is separated using fluorescence lifetime methods.
Fluorescence Imaging; Fluorescence Lifetime; Activatable Probe; Ratiometric
Simultaneous imaging of multiple cellular components is of tremendous importance in the study of complex biological systems, but the inability to use probes with similar emission spectra and the time consuming nature of collecting images on a confocal microscope are prohibitive. Hyperspectral imaging technology, originally developed for remote sensing applications, has been adapted to measure multiple genes in complex biological tissues. A spectral imaging microscope was used to acquire overlapping fluorescence emissions from specific mRNAs in brain tissue by scanning the samples using a single fluorescence excitation wavelength. The underlying component spectra obtained from the samples are then separated into their respective spectral signatures using multivariate analyses, enabling the simultaneous quantitative measurement of multiple genes either at regional or cellular levels.
hyperspectral imaging; immediate early genes; fluorescence imaging; pushbroom line-imaging; multivariate image analysis; spectral unmixing
Genetically encoded fluorescent indicators for bioimaging are powerful tools for visualizing biological phenomena in specified cell types or cellular compartments. However, available gene promoters or localization sequences are not applicable for visualizing all expression events. Furthermore, a visualization technique focusing on single cells or cellular compartments is required for characterizing specific cellular properties including individuality of cells in the cell population. To address these limitations, we developed a genetically encoded caged Ca2+ indicator for which expression timing and location could be controlled. This indicator, PA-TNXL, comprises a Ca2+-binding protein and troponin between a photoactivatable FRET donor (PA-GFP) and a FRET quencher (dim variant of YFP). Ultraviolet irradiation activates the FRET Ca2+ indicator. Using this indicator, we successfully imaged Ca2+ dynamics in a given set of HeLa cells and cultured hippocampal neurons. This technology can be applied for developing other photoactivatable indicators, thereby opening a new area of biological research.