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PubMed2004

A novel analytical approach for visualizing and tracking organic chemicals in plants.

Vegetation plays a key role in the environmental fate of many organic chemicals, from pesticides applied to plants, to the air-vegetation exchange and global cycling of atmospheric organic contaminants. Our ability to locate such compounds in plants has traditionally relied on inferences being made from destructive chemical extraction techniques or methods with potential artifacts. Here, for the first time, two-photon excitation microscopy (TPEM) is coupled with plant autofluorescence to visualize and track trace levels of an organic contaminant in living plant tissue, without any form of sample modification or manipulation. Anthracene-a polynuclear aromatic hydrocarbon (PAH)-was selected for study in living maize (Zea mays) leaves. Anthracene was tracked over 96 h, where amounts as low as approximately 0.1-10 pg were visible, as it moved through the epicuticular wax and plant cuticle, and was observed reaching the cytoplasm of the epidermal cells. By this stage, anthracene was identifiable in five separate locations within the leaf: (1) as a thin (approximately 5 microm) diffuse layer, in the upper surface of the epicuticular wax; (2) as thick (approximately 28 microm) diffuse bands extending from the epicuticular wax through the cuticle, to the cell walls of the epidermal cells; (3) on the external surface of epidermal cell walls; (4) on the internal surface of epidermal cell walls; and (5) within the cytoplasm of the epidermal cells. This technique provides a powerful nonintrusive tool for visualizing and tracking the movement, storage locations, and degradation of organic chemicals within vegetation using only plant and compound autofluorescence. Many other applications are envisaged for TPEM, in visualizing organic chemicals within different matrixes.

Air Pollutants↗
PubMed2004

ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.

Animals↗
PubMed2005

Water-soluble [2.2]paracyclophane chromophores with large two-photon action cross sections.

A series of alpha,omega-donor-substituted distyrylbenzene dimers held together by the [2.2]paracyclophane core were designed, synthesized, and characterized. Different substituents were chosen to modulate the strength of the donor nitrogen groups and to allow the molecules to be either neutral and soluble in nonpolar organic solvents or charged and water-soluble. The specific neutral structures are (in order of decreasing donor strength) 4,7,12,15-tetra[N,N-bis(6' '-chlorohexyl)-4'-aminostyryl]-[2.2]paracyclophane (1N), 4,7,12,15-tetra[(N-(6' '-chlorohexyl)carbazol-3'-yl)vinyl]-[2.2]paracyclophane (2N), and 4,7,12,15-tetra[N,N-bis(4' '-(6' ''-chlorohexyl)phenyl)-4'-aminostyryl]-[2.2]paracyclophane (3N). The charged species are 4,7,12,15-tetra[N,N-bis(6' '-(N,N,N-trimethylammonium)hexyl)-4'-aminostyryl]-[2.2]paracyclophane octaiodide (1C), 4,7,12,15-tetra[(N-(6' '-(N,N,N-trimethylammonium)hexyl)carbazol-3'-yl)vinyl]-[2.2]paracyclophane octaiodide (2C), and 4,7,12,15-tetra[N,N-bis(4' '-(6' ''-(N,N,N-trimethylammonium)hexyl)phenyl)-4'-aminostyryl]-[2.2]paracyclophane octaiodide (3C). Two-photon excitation spectra, measured using the two-photon induced fluorescence technique, show in toluene the following trend for the two-photon cross sections (delta): 3N > 2N > 1N. In water the delta values follow the same order, 3C approximately 2C > 1C, but are smaller (approximately one-third). Significantly, the fluorescence quantum yield (eta) in water decreases much more for 1, relative to 2 and 3. The two-photon action cross sections (deltaeta) of 2C and 3C are 294 GM and 359 GM, respectively. These values are among the highest reported thus far. These results show that, to maximize the deltaeta in this class of chromophores, one needs to fine-tune the magnitude of the charge transfer character of the excited state, to minimize fluorescence quenching in polar media.

Benzene Derivatives↗
PubMed2004

Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy.

Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled beta-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon beta-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes.

Cell Line↗
PubMed2005

Release and report: a new photolabile caging system with a two-photon fluorescence reporting function.

A new efficient photocaging system with a fluorescence reporting function has been developed. The photolabile latch is based on adducts of C-nucleophiles with aromatic ketones, such as thioxanthones and xanthones. The system is designed to quantify the release of biological effectors and to monitor their spatial distribution and localization by single- and two-photon fluorescence microscopy. In the armed state the ketone's conjugation is disrupted by nucleophilic addition, resulting in a blue shift of the absorption maxima and a dramatic decrease in fluorescence intensity. The mechanism of the photoinduced uncaging involves homolytic C-C bond fragmentation followed by radical disproportionation, regenerating the carbonyl moiety and restoring fluorescence. The uncaging can be initiated via either a one- or two-photon process, offering a new powerful tool for molecular life sciences. The synthesis and uncaging of dendrimer- and polymeric bead-based model systems are described.

Ketones↗
PubMed2005

Two-photon absorption in aqueous micellar solutions.

Addition of the surfactant sodium dodecyl sulfate at concentrations above the critical micelle concentration increases the fluorescence quantum yield and the two-photon absorption cross-section of charged [2.2]paracyclophane chromophores containing pairs of D-pi-D chromophores. The resulting spectra in the micellar solutions are very similar to those obtained for neutral isostructural analogues in toluene. The measured etadelta values are 1300 GM for 1C and 1920 GM for 2C, which are comparable or larger to those in toluene. These results highlight possible misleading interpretation of two-photon-induced emission for evaluating the concentration of labeled substrates used in two-photon microscopy and provide guidelines for designing molecular structures with optimized two-photon action cross-sections in water.

Acetates↗
PubMedDate not supplied

A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.

Riboflavin (vitamin B2, RF) is taken up in eukaryotic cells via specialized transport mechanisms. Although RF has fluorescence properties, direct microscopic visualization of RF uptake and trafficking has been complicated by cellular autofluorescence. We describe the synthesis, cellular uptake characteristics, and spectroscopic properties of a novel rhodamine-riboflavin conjugate (RD-RF), including absorption and emission spectra, two-photon excitation spectra, and fluorescence pH dependence. The conjugate has a molar extinction coefficient of 23 670 M(-1) cm(-1) at 545 nm (excitation wavelength) with a fluorescence quantum yield of 0.94. This compound exhibits intramolecular fluorescence resonance energy transfer (FRET). Selective quenching of the FRET signal is observed when RD-RF is bound with high affinity by the chicken riboflavin carrier protein. In addition to the typical rhodamine excitation and emission, FRET provides a secondary signal for conjugate localization and an in situ mechanism for observing riboflavin binding. Solution and in vitro stability determinations indicate that the linkage between riboflavin and rhodamine is stable for the duration of typical pulse--chase and cellular trafficking experiments. The distinct spectroscopic properties of RD-RF together with a comparable affinity for RF-binding proteins render it an excellent tool for the study of RF transport and trafficking in living cells.

Cell Compartmentation↗
PubMed2005

Two-photon photopolymerized tips for adhesion-free scanning-probe microscopy.

A hydrophobic polymeric tip of atomic force microscopy has been fabricated by two-photon adsorbed photopolymerization methods (TPAP). The fabrication was performed by a layer-by-layer polymerization of sliced multiple three-dimensional computer-aided design data. The used base resin was composed of acrylate and epoxy, which showed hydrophobic properties after the photopolymerization. For the sharp tip fabrication, we used a "dynamic partial polymerization method" which applied "the threshold effect" of TPAP to maximize the fabrication resolution. To investigate the performance of the fabricated polymeric tip, we have imaged several organic, inorganic, and biological samples using contact or dynamic force mode. The imaging results showed the hydrophobic polymeric tips solved various problems related to the tip adhesion to hydrophilic sample surfaces. Finally, the topographic image resolution of sub-5 nm was obtained using the polymeric tips for the hydrophilic mica surface.

Equipment Design↗
PubMed2006

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation--such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 degrees C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

Fluorescent Dyes↗
PubMed2006

Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.

Equipment Design↗
PubMed2006

Regulation of protein tyrosine phosphatase 1B by sumoylation.

Protein-tyrosine phosphatase 1B (PTP1B) is an ubiquitously expressed enzyme that negatively regulates growth-factor signalling and cell proliferation by binding to and dephosphorylating key receptor tyrosine kinases, such as the insulin receptor. It is unclear how the activity of PTP1B is regulated. Using a yeast two-hybrid assay, a protein inhibitor of activated STAT1 (PIAS1) was isolated as a PTP1B-interacting protein. Here, we show that PIAS1, which functions as a small ubiquitin-like modifier (SUMO) E3 ligase, associates with PTP1B in mammalian fibroblasts and catalyses sumoylation of PTP1B. Sumoylation of PTP1B reduces its catalytic activity and inhibits the negative effect of PTP1B on insulin receptor signalling and on transformation by the oncogene v-crk. Insulin-stimulated sumoylation of endogenous PTP1B results in a transient downregulation of the enzyme; this event does not occur when the endogenous enzyme is replaced with a sumoylation-resistant mutant of PTP1B. These results suggest that sumoylation, which has been implicated primarily in processes in the nucleus and nuclear pore, also modulates a key enzyme-substrate signalling complex that regulates metabolism and cell proliferation.

Animals↗
PubMed2004

Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo.

Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.

Animals↗
PubMed2004

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells.

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

Animals↗
PubMed2006

Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy.

Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1-2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with approximately 1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues.

Animals↗