Advances in the chemistry of small molecule fluorescent probes

Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes.     

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their ⑴broad absorption spectra allowing visualization with single light source, ⑵exceptional photo-stability allowing long term studies and ⑶narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.     

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (~7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ~8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.     

Fluorescence Tracing of Intracellular Proteins

Developments in fluorescence microscopy and the availability of fluorescently labeled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci allowing deep insight into cell and organelle biology. Furthermore, confocal microscopy permits analysis of the three dimensional architecture of cells that could not be accomplished by conventional light microscopy. The goal of fluorescence protein tracing by microscopy is to visualize cellular constituents and general cytoarchitecture as close to native organization as possible. To achieve this, and to preserve cellular structure in the best possible manner, the specimen is usually fixed chemically. Here I review several standard fixation, permeabilization and labeling schemes followed by examples of several standard imaging techniques.     

Spectrofluorimetric methods for the measurement of the so called ”Microviscosity“ of lipidic structures

The cellular interactions involving the membrane depend on its physico-chemical nature and on the topographical distribution of the membrane receptors. At present, the role of the lipidic regions is not well defined; however, it is known that the fluidity or « microviscosityof the lipidic components controls important processes in cellular biology.Different spectrofluorimetric methods, continuous or time resolved, susceptible to the cohesion of lipidic regions have been developed:

1. The anisotropy of fluorescence where the rotation of probes is studied (linked to the coefficient of diffusional rotation).

2. The inhibition of fluorescence where the kinetics of the reaction is practically diffusion controlled.

3. The formation of emissive intramolecular complexes where the internal rotations of interchromophoric bonds are studied.

After the development of kinetic models, the methods have been tested with synthetic and natural organized assemblies.The values of « microviscosityobtained with these methods may be different because the environments of probes are different. Therefore, the concept of « microviscosity , applied to biological membranes is limited.     

Fluorescence tools to investigate riboswitch structural dynamics

Riboswitches are novel regulatory elements that respond to cellular metabolites to control gene expression. They are constituted of highly conserved domains that have evolved to recognize specific metabolites. Such domains, so-called aptamers, are folded into intricate structures to enable metabolite recognition. Over the years, the development of ensemble and single-molecule fluorescence techniques has allowed to probe most of the mechanistic aspects of aptamer folding and ligand binding. In this review, we summarize the current fluorescence toolkit available to study riboswitch structural dynamics. We fist describe those methods based on fluorescent nucleotide analogues, mostly 2-aminopurine (2AP), to investigate short-range conformational changes, including some key steady-state and time-resolved examples that exemplify the versatility of fluorescent analogues as structural probes. The study of long-range structural changes by Förster resonance energy transfer (FRET) is mostly discussed in the context of single-molecule studies, including some recent developments based on the combination of single-molecule FRET techniques with controlled chemical denaturation methods. This article is part of a Special Issue entitled: Riboswitches.     

Luminescent metal-ligand complexes as probes of macromolecular interactions and biopolymer dynamics

The knowledge of microsecond dynamics is important for an understanding of the mechanism and function of biological systems. Fluorescent techniques are well established in biophysical studies, but their applicability to probe microsecond timescale processes is limited. Luminescent metal-ligand complexes (MLCs) have created interest mainly due to their unique luminescent properties, such as the exceptionally long decay times and large fundamental anisotropy values, allowing examination of microsecond dynamics by fluorescence methods. MLC properties also greatly simplify instrumentation requirements and enable the use of light emitting diode excitation for time-resolved measurements. Recent literature illustrates how MLC labels take full advantage of well developed fluorescence techniques and how those methods can be extended to timescales not easily accessible with nanosecond probes. MLCs are now commercially available as reactive labels which give researchers access to methods that previously required more complex approaches. The present paper gives an overview of the applications of MLC probes to studies of molecular dynamics and interactions of proteins, membranes and nucleic acids.     

Structural and functional imaging with carbon nanotube AFM probes

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography, chemically functionalized AFM probes can measure the spatial arrangement of chemical functional groups in a sample. However, standard silicon and silicon nitride tips, once functionalized, do not yield sufficient resolution to allow combined structural and functional imaging of biomolecules. The unique end-group chemistry of carbon nanotubes, which can be arbitrarily modified by established chemical methods, has been exploited for chemical force microscopy, allowing single-molecule measurements with well-defined functionalized tips.     

The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells

Background

Small molecule fluorescent probes are vital tools for monitoring reactive oxygen species in cells.

Scope of review

The types of probe available, the extent to which they are specific or quantitative and complications in interpreting results are discussed.

Major conclusions

Most commonly used probes (e.g. dihydrodichlorofluorescein, dihydrorhodamine) have some value in providing information on changes to the redox environment of the cell, but they are not specific for any one oxidant and the response is affected by numerous chemical interactions and not just increased oxidant generation. These probes generate the fluorescent end product by a free radical mechanism, and to react with hydrogen peroxide they require a metal catalyst. Probe radicals can react with oxygen, superoxide, and various antioxidant molecules, all of which influence the signal. Newer generation probes such as boronates act by a different mechanism in which nucleophilic attack by the oxidant on a blocking group releases masked fluorescence. Boronates react with hydrogen peroxide, peroxynitrite, hypochlorous acid and in some cases superoxide, so are selective but not specific. They react with hydrogen peroxide very slowly, and kinetic considerations raise questions about how the reaction could occur in cells.

General significance

Data from oxidant-sensitive fluorescent probes can provide some information on cellular redox activity but is widely misinterpreted. Recently developed non-redox probes show promise but are not generally available and more information on specificity and cellular reactions is needed. We do not yet have probes that can quantify cellular production of specific oxidants. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

The labile iron pool: characterization,measurement, and participation in cellular processes(1)

The cellular labile iron pool (LIP) is a pool of chelatable and redox-active iron, which is transitory and serves as a crossroad of cell iron metabolism. Various attempts have been made to analyze the levels of LIP following cell disruption. The chemical identity of this pool has remained poorly characterized due to the multiplicity of iron ligands present in cells. However, the levels of LIP recently have been assessed with novel nondisruptive techniques that rely on the application of fluorescent metalosensors. Methodologically, a fluorescent chelator loaded into living cells binds to components of the LIP and undergoes stoichiometric fluorescence quenching. The latter is revealed and quantified in situ by addition of strong permeating iron chelators. Depending on the intracellular distribution of the sensing and chelating probes, LIP can be differentially traced in subcellular structures, allowing the dynamic assessment of its levels and roles in specific cell compartments. The labile nature of LIP was also revealed by its capacity to promote formation of reactive oxygen species (ROS), whether from endogenous or exogenous redox-active sources. LIP and ROS levels were shown to follow similar "rise and fall" patterns as a result of changes in iron import vs. iron chelation or ferritin (FT) degradation vs. ferritin synthesis. Those patterns conform with the accepted role of LIP as a self-regulatory pool that is sensed by cytosolic iron regulatory proteins (IRPs) and feedback regulated by IRP-dependent expression of iron import and storage machineries. However, LIP can also be modulated by biochemical mechanisms that override the IRP regulatory loops and, thereby, contribute to basic cellular functions. This review deals with novel methodologies for assessing cellular LIP and with recent studies in which changes in LIP and ROS levels played a determining role in cellular processes.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.     

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).     

Zeta potential mediated reaction monitoring on nano and microparticles

Nano and microparticles are widely used across the life science interface, with applications ranging from chemical probes of biological function to fluorescent particles for flow cytometry and cellular tracking. Increasingly, particles are modified with a variety of chemistries to boost their functionality and broaden their biological applicability. However, although particle modification has become standard laboratory practice, the ability to determine the extent and efficiency of chemical modification is often very limited and empirical in nature. Herein, we report the use of zeta potential analysis as a simple and rapid "direct-on-particle" approach allowing levels of bead modification and derivatization to be evaluated. As a proof-of-concept, aminomethyl-functionalized nano and microparticles were derivatized to display a variety of surface functionalities and their zeta potentials measured, allowing verification of the applicability of the approach for particle analysis. We demonstrate that zeta potential measurement is a convenient approach which allows multistep reaction sequences to be followed, and show that this method can be used to verify and validate successful particle modification.     

Coupling chemical biology and vibrational spectroscopy for studies of amyloids in vitro and in cells

Amyloid diseases are characterized by the aggregation of various proteins to form insoluble β-sheet–rich fibrils leading to cell death. Vibrational spectroscopies have emerged as attractive methods to study this process because of the rich structural information that can be extracted without large, perturbative probes. Importantly, specific vibrations such as the amide-I band directly report on secondary structure changes, which are key features of amyloid formation. Beyond intrinsic vibrations, the incorporation of unnatural vibrational probes can improve sensitivity for secondary structure determination (e.g. isotopic labeling), can provide residue-specific information of the surrounding polarity (e.g. unnatural amino acid), and are translatable into cellular studies. Here, we review the latest studies that have leveraged tools from chemical biology for the incorporation of novel vibrational probes into amyloidogenic proteins for both mechanistic and cellular studies.     

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects

Chemical probes for free radicals in biology are important tools; fluorescence and chemiluminescence offer high detection sensitivity. This article reviews progress in the development of probes for "reactive oxygen and nitrogen" species, emphasizing the caution needed in their use. Reactive species include hydrogen peroxide; hydroxyl, superoxide, and thiyl radicals; carbonate radical-anion; and nitric oxide, nitrogen dioxide, and peroxynitrite. Probes based on reduced dyes lack selectivity and may require a catalyst for reaction: despite these drawbacks, dichlorodihydrofluorescein and dihydrorhodamine have been used in well over 2,000 studies. Use in cellular systems requires loading into cells, and minimizing leakage. Reactive species can compete with intracellular antioxidants, changes in fluorescence or luminescence possibly reflecting changes in competing antioxidants rather than free radical generation rate. Products being measured can react further with radicals, and intermediate probe radicals are often reactive toward antioxidants and especially oxygen, to generate superoxide. Common probes for superoxide and nitric oxide require activation to a reactive intermediate; activation is not achieved by the radical of interest and the response is thus additionally sensitive to this first step. Rational use of probes requires understanding and quantitation of the mechanistic pathways involved, and of environmental factors such as oxygen and pH. We can build on this framework of knowledge in evaluating new probes.     

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ～20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.     

应用荧光分析法检测酶的研究进展

酶是细胞新陈代谢的基础,酶的检测在生物技术、疾病诊断及药物开发等领域都具有十分重要的意义。在检测酶的诸多方法中,荧光法因其灵敏度高、检测限低等优势发展迅速。以下简述了近年来荧光法在酶检测领域的研究,根据检测方法的不同分为直接荧光检测法和间接荧光检测法,其中直接检测法又根据不同的底物标记及检测机理进行分类。以下介绍了各种方法的应用,并展望了此类方法的前景和发展趋势,为酶工程及生命科学其他领域的相关研究提供信息。     

On the nature of the energised state of submitochondrial particles; investigations with N-aryl naphthalene sulphonate probes.

1. A further investigation has been made of the way in which the fluorescent probes 1-anilino-naphthalene-8-sulphonate and 2-(N-methyl-anilino) naphthalene-6-sulphonate report on the energised state of bovine heart submitochondrial particles. 2. A comparison of the probe responses to energisation with ATP or to a potassium diffusion potential has been made. The fluorescence enhancements seen in these two cases have different characteristics, and in view of this it is questioned whether a substrate generated energised state of a submitochondrial particle can be equated with a trans-membrane potassium diffusion potential. 3. Substitution of ITP for ATP reduces the rate at which either of the probes respond to energisation. In contrast reducing the ATPase activity of the particles by treatment with the covalent ATPase inhibitors 4-chloro-7-nitrobenzofurazan or N,N'-dicyclohexyl-carbodiimide has no effect on this rate. This finding that the rate of the fluorescence changes is directly sensitive to events at the level of the ATPase, but not to the total ATPase activity, suggests that this rate may not be controlled by a delocalised energised state. Reduction of ATPase activity decreases the extent of the fluorescence enhancement and a relationship between the change in probe fluorescence and ATPase activity is given. 4. The results in this paper are discussed in the context of the mechanisms which have been proposed to account for the fluorescence enhancements of N-aryl naphthalene sulphonate probes upon energisation of submitochondrial particles.     

Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.     

Chemical probes for analysis of carbonylated proteins: A review

Protein carbonylation is a major form of protein oxidation and is widely used as an indicator of oxidative stress. Carbonyl groups do not have distinguishing UV or visible, spectrophotometric absorbance/fluorescence characteristics and thus their detection and quantification can only be achieved using specific chemical probes. In this paper, we review the advantages and disadvantages of several chemical probes that have been and are still being used for protein carbonyl analysis. These probes include 2,4-dinitrophenylhydazine (DNPH), tritiated sodium borohydride ([³H]NaBH₄), biotin-containing probes, and fluorescence probes. As our discussions lean toward gel-based approaches, utilizations of these probes in 2D gel-based proteomic analysis of carbonylated proteins are illustrated where applicable. Analysis of carbonylated proteins by ELISA, immunofluorescent imaging, near infrared fluorescence detection, and gel-free proteomic approaches are also discussed where appropriate. Additionally, potential applications of blue native gel electrophoresis as a tool for first dimensional separation in 2D gel-based analysis of carbonylated proteins are discussed as well.