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Journal articles on the topic 'Orientation of fluorescent probes'

Author: Grafiati

Published: 4 June 2021

Last updated: 18 February 2022

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1

Nakai, Nori, Keisuke Sato, Tomomi Tani, Kenta Saito, Fumiya Sato, and Sumio Terada. "Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy." Microscopy 68, no. 5 (July 2, 2019): 359–68. http://dx.doi.org/10.1093/jmicro/dfz022.

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Abstract Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.

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2

Sugizaki, Ayana, Keisuke Sato, Kazuyoshi Chiba, Kenta Saito, Masahiko Kawagishi, Yuri Tomabechi, Shalin B. Mehta, et al. "POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos." Proceedings of the National Academy of Sciences 118, no. 11 (March 5, 2021): e2019071118. http://dx.doi.org/10.1073/pnas.2019071118.

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Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISactwith FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

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3

Filipe, Hugo A. L., Maria João Moreno, and Luís M. S. Loura. "The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations." Molecules 25, no. 15 (July 28, 2020): 3424. http://dx.doi.org/10.3390/molecules25153424.

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Fluorescent probes have been employed for more than half a century to study the structure and dynamics of model and biological membranes, using spectroscopic and/or microscopic experimental approaches. While their utilization has led to tremendous progress in our knowledge of membrane biophysics and physiology, in some respects the behavior of bilayer-inserted membrane probes has long remained inscrutable. The location, orientation and interaction of fluorophores with lipid and/or water molecules are often not well known, and they are crucial for understanding what the probe is actually reporting. Moreover, because the probe is an extraneous inclusion, it may perturb the properties of the host membrane system, altering the very properties it is supposed to measure. For these reasons, the need for independent methodologies to assess the behavior of bilayer-inserted fluorescence probes has been recognized for a long time. Because of recent improvements in computational tools, molecular dynamics (MD) simulations have become a popular means of obtaining this important information. The present review addresses MD studies of all major classes of fluorescent membrane probes, focusing in the period between 2011 and 2020, during which such work has undergone a dramatic surge in both the number of studies and the variety of probes and properties accessed.

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4

Husain, Ali, Asaithampi Ganesan, Miloslav Machacek, Lukas Cerveny, Pavel Kubat, Basma Ghazal, Petr Zimcik, and Saad Makhseed. "Dually directional glycosylated phthalocyanines as extracellular red-emitting fluorescent probes." Dalton Transactions 49, no. 28 (2020): 9605–17. http://dx.doi.org/10.1039/d0dt01180k.

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Control of the spatial orientation of glycosylated peripheral substituents in phthalocyanines provides monomeric species that are highly fluorescent in water. Due to their hydrophilic nature, they are suitable as extracellular fluorescent probes.

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5

Czajkowsky, Daniel M. "Fluorescence anisotropy of oligomeric proteins." Spectroscopy 18, no. 1 (2004): 85–93. http://dx.doi.org/10.1155/2004/460353.

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Previous studies of protein oligomerization using time-resolved fluorescence anisotropy assumed a single fixed probe per oligomeric complex and an identical probe orientation in complexes of different stoichiometry. However, an oligomer consisting of “n” singly labeled monomers must necessarily have “n” probes. Moreover, in the expression for the anisotropy decay, the molecular axes from which the probe orientation is defined are different for complexes that differ in stoichiometry. Here, we derive an expression for the decay of the anisotropy for molecules with any number of fixed probes, and show how an explicit understanding of the probe orientation is necessary to properly assess oligomerization.

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6

Dale, R. E., S. C. Hopkins, U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman. "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers." Biophysical Journal 76, no. 3 (March 1999): 1606–18. http://dx.doi.org/10.1016/s0006-3495(99)77320-0.

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7

Filipe, Hugo A. L., Šárka Pokorná, Martin Hof, Mariana Amaro, and Luís M. S. Loura. "Orientation of nitro-group governs the fluorescence lifetime of nitrobenzoxadiazole (NBD)-labeled lipids in lipid bilayers." Physical Chemistry Chemical Physics 21, no. 4 (2019): 1682–88. http://dx.doi.org/10.1039/c8cp06064a.

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NBD fluorescence lifetime varies significantly from one lipid probe to another, despite identical fluorophore locations in the membrane. This is a consequence of differences among probes in the orientation of NBD, which determines the exposure to water of the NO₂ group.

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8

Ling, N., C. Shrimpton, J. Sleep, J. Kendrick-Jones, and M. Irving. "Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle." Biophysical Journal 70, no. 4 (April 1996): 1836–46. http://dx.doi.org/10.1016/s0006-3495(96)79749-7.

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9

Roorda, Robert D., Tobias M. Hohl, Ricardo Toledo-Crow, and Gero Miesenböck. "Video-Rate Nonlinear Microscopy of Neuronal Membrane Dynamics With Genetically Encoded Probes." Journal of Neurophysiology 92, no. 1 (July 2004): 609–21. http://dx.doi.org/10.1152/jn.00087.2004.

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Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.

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10

Backer, Adam S., Andreas S. Biebricher, Graeme A. King, Gijs J. L. Wuite, Iddo Heller, and Erwin J. G. Peterman. "Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA." Science Advances 5, no. 3 (March 2019): eaav1083. http://dx.doi.org/10.1126/sciadv.aav1083.

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DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology such as programmable materials and DNA origami. While some features of DNA conformational changes are well characterized, fundamental information such as the orientations of the DNA base pairs is unknown. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of S-DNA, an elusive, elongated conformation that can be accessed by mechanical overstretching. To this end, we directly quantify the orientations and rotational dynamics of fluorescent DNA-intercalated dyes. At extensions beyond the DNA overstretching transition, intercalators adopt a tilted (θ ~ 54°) orientation relative to the DNA axis, distinct from the nearly perpendicular orientation (θ ~ 90°) normally assumed at lower extensions. These results provide the first experimental evidence that S-DNA has substantially inclined base pairs relative to those of the standard (Watson-Crick) B-DNA conformation.

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11

Licari, Giuseppe, Karolina Strakova, Stefan Matile, and Emad Tajkhorshid. "Twisting and tilting of a mechanosensitive molecular probe detects order in membranes." Chemical Science 11, no. 22 (2020): 5637–49. http://dx.doi.org/10.1039/d0sc02175j.

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12

Sabido-David, Cibele, Seth C. Hopkins, Lakshmi D. Saraswat, Susan Lowey, Yale E. Goldman, and Malcolm Irving. "Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres." Journal of Molecular Biology 279, no. 2 (June 1998): 387–402. http://dx.doi.org/10.1006/jmbi.1998.1771.

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13

Klymchenko, Andrey S., Guy Duportail, Turan Ozturk, Vasyl G. Pivovarenko, Yves Mély, and Alexander P. Demchenko. "Novel Two-Band Ratiometric Fluorescence Probes with Different Location and Orientation in Phospholipid Membranes." Chemistry & Biology 9, no. 11 (November 2002): 1199–208. http://dx.doi.org/10.1016/s1074-5521(02)00244-2.

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14

Soleimaninejad, Hamid, Kenneth P. Ghiggino, Trevor A. Smith, and Matthew F. Paige. "Fluorescence anisotropy imaging of a polydiacetylene photopolymer film." Canadian Journal of Chemistry 97, no. 6 (June 2019): 422–29. http://dx.doi.org/10.1139/cjc-2018-0360.

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UV-illumination of phase-separated surfactant films prepared from mixtures of photopolymerizable 10,12-pentacosadiynoic acid and perfluorotetradecanoic acid results in the formation of fluorescent polydiacetylene fibers and aggregates. In this work, the orientation of polymer strands that comprise the resulting photopolymer structures has been probed using fluorescence anisotropy imaging in combination with defocused single-molecule fluorescence imaging. Imaging experiments indicate the presence of significant fiber-to-fiber heterogeneity, as well as anisotropy within each fiber (or aggregate), with both of these properties changing as a function of film preparation conditions. This anisotropy can be attributed to various alignments of the constituent polymer strands that comprise the larger fibers and aggregates. Intriguingly, when using defocused imaging, fiber images consisted of a series of discrete “doughnut” fluorescence emission patterns, which exhibited intermittent on–off blinking behavior; both of these properties are characteristic of individual emission transition dipoles (single molecules). Further, all of the individual emission transition dipoles had a uniform orientation with respect to the axis of the fiber, indicating a common orientation of discrete emitters in the larger polymer fiber. The implications of these results for future studies of the electronic properties of conjugated polymers in larger macroscopic systems are noted.

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15

Weißenstein, Annike, Myroslav O. Vysotsky, Ivo Piantanida, and Frank Würthner. "Naphthalene diimide–amino acid conjugates as novel fluorimetric and CD probes for differentiation between ds-DNA and ds-RNA." Beilstein Journal of Organic Chemistry 16 (August 19, 2020): 2032–45. http://dx.doi.org/10.3762/bjoc.16.170.

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Two novel unnatural amino acids, prepared by linking a dicationic purple-coloured and fluorescent naphthalene diimide (NDI) at core position to amino acid side chains of variable length, strongly interacted with ds-DNA/RNA by threading intercalation. Different from a reference NDI dye with identical visible range absorbance (520–540 nm) and Stokes shifts in emission (+60 nm, quantum yield > 0.2), only these amino acid–NDI conjugates showed selective fluorimetric response for GC-DNA in respect to AT(U)-polynucleotides. The DNA/RNA binding-induced circular dichroism (ICD) response of NDI at 450–550 nm strongly depended on the length and rigidity of the linker to the amino acid unit, which controls the orientation of the NDI unit inside within the intercalative binding site. The ICD selectivity also depends on the type of polynucleotide, thus the studied NDI dyes act as dual fluorimetric/ICD probes for sensing the difference between here used GC-DNA, AT-DNA and AU-RNA.

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16

Levitus, Marcia, and Suman Ranjit. "Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments." Quarterly Reviews of Biophysics 44, no. 1 (November 26, 2010): 123–51. http://dx.doi.org/10.1017/s0033583510000247.

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AbstractThe breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis–trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

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17

Wasif Baig, Mirza, Marek Pederzoli, Piotr Jurkiewicz, Lukasz Cwiklik, and Jiri Pittner. "Orientation of Laurdan in Phospholipid Bilayers Influences Its Fluorescence: Quantum Mechanics and Classical Molecular Dynamics Study." Molecules 23, no. 7 (July 13, 2018): 1707. http://dx.doi.org/10.3390/molecules23071707.

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Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid–crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid–crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.

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18

Quesada, Ernesto, Malin Ardhammar, Bengt Nordén, Michel Miesch, Guy Duportail, Yvonne Bonzi-Coulibaly, Yoichí Nakatani, and Guy Ourisson. "Synthesis and Fluorescence Properties of Novel Transmembrane Probes and Determination of Their Orientation within Vesicles." Helvetica Chimica Acta 83, no. 9 (September 6, 2000): 2464–76. http://dx.doi.org/10.1002/1522-2675(20000906)83:9<2464::aid-hlca2464>3.0.co;2-t.

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19

Lewis, John H., John F. Beausang, H. Lee Sweeney, and Yale E. Goldman. "The azimuthal path of myosin V and its dependence on lever-arm length." Journal of General Physiology 139, no. 2 (January 30, 2012): 101–20. http://dx.doi.org/10.1085/jgp.201110715.

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Myosin V (myoV) is a two-headed myosin capable of taking many successive steps along actin per diffusional encounter, enabling it to transport vesicular and ribonucleoprotein cargos in the dense and complex environment within cells. To better understand how myoV navigates along actin, we used polarized total internal reflection fluorescence microscopy to examine angular changes of bifunctional rhodamine probes on the lever arms of single myoV molecules in vitro. With a newly developed analysis technique, the rotational motions of the lever arm and the local orientation of each probe relative to the lever arm were estimated from the probe’s measured orientation. This type of analysis could be applied to similar studies on other motor proteins, as well as other proteins with domains that undergo significant rotational motions. The experiments were performed on recombinant constructs of myoV that had either the native-length (six IQ motifs and calmodulins [CaMs]) or truncated (four IQ motifs and CaMs) lever arms. Native-length myoV-6IQ mainly took straight steps along actin, with occasional small azimuthal tilts around the actin filament. Truncated myoV-4IQ showed an increased frequency of azimuthal steps, but the magnitudes of these steps were nearly identical to those of myoV-6IQ. The results show that the azimuthal deflections of myoV on actin are more common for the truncated lever arm, but the range of these deflections is relatively independent of its lever-arm length.

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20

Karlsson, Martin, and Uno Carlsson. "Protein Adsorption Orientation in the Light of Fluorescent Probes: Mapping of the Interaction between Site-Directly Labeled Human Carbonic Anhydrase II and Silica Nanoparticles." Biophysical Journal 88, no. 5 (May 2005): 3536–44. http://dx.doi.org/10.1529/biophysj.104.054809.

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21

Cupane, A., A. Dembska, T. Pedzinski, S. Takenaka, and B. Juskowiak. "Emission lifetime study of fluorescence probes based on G-quadruplex oligonucleotides end-labeled with pyrene moieties." Spectroscopy 24, no. 3-4 (2010): 325–31. http://dx.doi.org/10.1155/2010/591086.

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Fluorescence lifetime study of two probes abbreviated as Py-Htelom-Py and Py-TBA-Py, carrying pyrene moieties at both termini and sequences of Human telomere and Thrombin Binding Aptamer, respectively are reported. The effect of potassium ion on the photophysical processes was examined in order to elucidate factors that facilitate the production of excimer emission. Emission kinetics data indicated that the relative orientation of pyrene and neighboring nucleobase (guanine, adenine, thymine) plays a crucial role in determining both the rate of electron-transfer quenching of pyrene excited state and the efficiency of excimer emission.

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22

Wang, Yu-li. "Fluorescence microscopy of molecular organization and dynamics in cultured cells." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (August 1992): 550–51. http://dx.doi.org/10.1017/s0424820100123155.

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Over the past ten years various technical advances have allowed the direct study of molecular activities in cultured cells under a fluorescence microscope. Fluorescent probes are well known for their high sensitivity, specificity and amenability to various spectroscopic analyses. When used in conjunction with low-light-level detectors and image processing computers, high resolution images of weak signals from single cells can be successfully acquired. In addition, the availability of digitized images has greatly facilitated the extraction of photometric and morphometric information.We use Zeiss inverted microscopes equipped with epi-illuminators and Dage-MTI ISIT video cameras or Photometrics cooled CCD cameras. Custom incubator systems built on the microscope stages allow the maintenance of live cells for up to several days. The signals are processed with image processing systems (Imaging Technologies) interfaced with graphics workstations (Silicon Graphics, Model 3130 or 4D/20) or personal computers (386/33). All images are acquired by frame averaging/signal integration, followed by subtraction of the dark noise, and storage as computer files. A variation of this simple processing strategy has allowed the detection of extremely weak signals that are essentially invisible on unprocessed ISIT images. Computer programs are then used to display sequences or images as motion pictures, to measure the linear dimension and angular orientation, and to integrate intensities over defined areas.

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23

Komuro, Shirabe, Ryota Endo, Kaori Shikata, and Akio Kato. "Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure." Genome 56, no. 3 (March 2013): 131–37. http://dx.doi.org/10.1139/gen-2013-0003.

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Wheat (Triticum aestivum L.) is an allohexaploid, in which each of the three genomes has a high 1C content. This indicates the presence of multiple tandemly repeated sequences, which should be detectable using in situ hybridization. Some repeats have already been described, but others remain to be recognized. To discover others, 2000 plasmid wheat clones were examined for signal presence after fluorescence in situ hybridization and microscopic signal observation. Among them, 47 clones produced strong discrete signals on wheat chromosomes. Two of the newly identified clones (pTa-535 and pTa-713) were determined to have especially valuable sequences for chromosome identification. In combination with pTa-86 (the pSc119 homologous sequence), these probes enable unambiguous discrimination of all wheat chromosomes including orientation. Four newly identified sequences (pTa-465, pTa-k566, pTa-s120, and pTa-s126) were useful in that they produced discrete signals on various wheat chromosome arms. Two other clones (pTa-k288 and pTa-k229) produced GISH-like (genomic in situ hybridization) signals because they allowed the A, B, and D genomes to be distinguished simultaneously. In addition, centromere, centromere-related, and ribosomal DNA clones were identified. Also described are improvements on slide preparation and reprobing procedures. To enhance discrete signal detection, a new direct fluorescent-labeling procedure, namely the VentR (exo-) terminal extension method, was employed.

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24

Larson, D. M., R. J. Gilbert, and E. C. Beyer. "Two-dimensional coupling by gap junctions in cultured gastric smooth muscle monolayers." American Journal of Physiology-Gastrointestinal and Liver Physiology 263, no. 2 (August 1, 1992): G261—G268. http://dx.doi.org/10.1152/ajpgi.1992.263.2.g261.

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We studied intercellular transfer in cultured rabbit gastric smooth muscle cell monolayers after microinjection of electrotonic current or the fluorescent probe Lucifer yellow CH. Because cultured gastric muscle cells proliferate in vitro and form regular arrays of parallel spindle-shaped cells, we sought to assess the role of cell shape and orientation in determining two-dimensional coupling properties. With the use of electron microscopy, gap junctions were identified between adjacent cells. Northern blot analyses using specific cDNA probes demonstrated expression of mRNA for the gap junction protein connexin43. Dye injection of Lucifer yellow resulted in 97% transfer to at least one adjacent cell, and 88% of adjacent cells received dye. Electrophysiological studies were performed using two intracellular microelectrodes to measure electrotonic current flow between cells at varying interelectrode distances. Current flow in the monolayers was modeled using a modified two-dimensional analysis. Initial assessment showed that the ratio of calculated space constants (longitudinal axis/perpendicular axis) was 4.4, indicating anisotropic conditions. However, when a geometric transform was used to normalize the spindle-shaped cells to regular hexagons, the space constants became statistically equivalent (200 microns longitudinal, 256 microns perpendicular). These results suggest that anisotropy of current flow in the monolayer of gastric smooth muscle cells was due primarily to the shape of the cells and not to intrinsic membrane properties or the distribution of gap junctions.

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25

Ice, G. E. "X-ray microprobe for materials science." Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 56–57. http://dx.doi.org/10.1017/s0424820100168013.

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The increasing availability of synchrotron x-ray sources has stimulated the development of advanced hard x-ray (E≥5 keV) microprobes. With new x-ray optics these microprobes can achieve micron and submicron spatial resolutions. The inherent elemental and crystallographic sensitivity of an x-ray microprobe and its inherently nondestructive and penetrating nature will have important applications to materials science. For example, x-ray fluorescent microanalysis of materials can reveal elemental distributions with greater sensitivity than alternative nondestructive probes. In materials, segregation and nonuniform distributions are the rule rather than the exception. Common interfaces to whichsegregation occurs are surfaces, grain and precipitate boundaries, dislocations, and surfaces formed by defects such as vacancy and interstitial configurations. In addition to chemical information, an x-ray diffraction microprobe can reveal the local structure of a material by detecting its phase, crystallographic orientation and strain.Demonstration experiments have already exploited the penetrating nature of an x-ray microprobe and its inherent elemental sensitivity to provide new information about elemental distributions in novel materials.

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26

Kim, Jeong-Soon, Kevin L. Childs, M. Nurul Islam-Faridi, Monica A. Menz, Robert R. Klein, Patricia E. Klein, H. James Price, John E. Mullet, and David M. Stelly. "Integrated karyotyping of sorghum by in situ hybridization of landed BACs." Genome 45, no. 2 (April 1, 2002): 402–12. http://dx.doi.org/10.1139/g01-141.

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The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.Key words: integrated karyotyping, FISH, sorghum, BAC.

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27

Kinosita, Kazuhiko, Ryohei Yasuda, Ichiro Sase, and Hidetake Miyata. "Attempts to Visualize Conformational Changes in a Single Protein Molecule During Function." Microscopy and Microanalysis 3, S2 (August 1997): 799–800. http://dx.doi.org/10.1017/s1431927600010886.

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The operation of protein molecular machines is essentially stochastic. One cannot predict exactly when an ion channel opens, or when a molecular motor makes a step. As such, working molecular machines cannot be synchronized with each other in a rigorous sense. To understand the mechanism of a protein machine, therefore, one has to watch conformational changes of individual molecules while they are at work. To this end, we have been trying to develop two approaches based on optical microscopy. One is to attach a small tag, a fluorescent probe, to an appropriate site on the protein molecule of interest. Polarization of the probe fluorescence, for example, will reveal the orientation of the fluorophore, and thus of the portion of the protein molecule to which the fluorophore is attached. A conformational change of the protein will be detected as the reorientation of the fluorophore. A prerequisite is the ability to image a single fluorophore in an aqueous environment, which we have achieved on an epifluorescence microscope by reducing its background luminescence by two orders of magnitude

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Catalano, D., A. Lenzi, and C. A. Veracini. "Orientational order and dynamics of fluorescent probes in aligned lyotropic phases by fluorescence depolarization spectroscopy. Perylene in a nematic discotic phase. A comparison with2H NMR." Liquid Crystals 14, no. 5 (January 1993): 1457–68. http://dx.doi.org/10.1080/02678299308026458.

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Vozdova, Miluse, Svatava Kubickova, Halina Cernohorska, Jan Fröhlich, and Jiri Rubes. "Anchoring the CerEla1.0 Genome Assembly to Red Deer (Cervus elaphus) and Cattle (Bos taurus) Chromosomes and Specification of Evolutionary Chromosome Rearrangements in Cervidae." Animals 11, no. 9 (September 6, 2021): 2614. http://dx.doi.org/10.3390/ani11092614.

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The family Cervidae groups a range of species with an increasing economic significance. Their karyotypes share 35 evolutionary conserved chromosomal segments with cattle (Bos taurus). Recent publication of the annotated red deer (Cervus elaphus) whole genome assembly (CerEla1.0) has provided a basis for advanced genetic studies. In this study, we compared the red deer CerEla1.0 and bovine ARS-UCD1.2 genome assembly and used fluorescence in situ hybridization with bovine BAC probes to verify the homology between bovine and deer chromosomes, determined the centromere-telomere orientation of the CerEla1.0 C-scaffolds and specified positions of the cervid evolutionary chromosome breakpoints. In addition, we revealed several incongruences between the current deer and bovine genome assemblies that were shown to be caused by errors in the CerEla1.0 assembly. Finally, we verified the centromere-to-centromere orientation of evolutionarily fused chromosomes in seven additional deer species, giving a support to previous studies on their chromosome evolution.

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Voglino, L., S. A. Simon, and T. J. McIntosh. "Orientation of LamB Signal Peptides in Bilayers: Influence of Lipid Probes on Peptide Binding and Interpretation of Fluorescence Quenching Data†." Biochemistry 38, no. 23 (June 1999): 7509–16. http://dx.doi.org/10.1021/bi990099q.

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Quesada, Ernesto, Malin Ardhammar, Bengt Norden, Michel Miesch, Guy Duportail, Yvonne Bonzi-Coulibaly, Yoichi Nakatani, and Guy Ourisson. "ChemInform Abstract: Synthesis and Fluorescence Properties of Novel Transmembrane Probes (I) and (II) and Determination of Their Orientation within Vesicles." ChemInform 31, no. 51 (December 19, 2000): no. http://dx.doi.org/10.1002/chin.200051103.

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32

Nagele, R. G., T. Freeman, L. McMorrow, Z. Thomson, K. Kitson-Wind, and Hy Lee. "Chromosomes exhibit preferential positioning in nuclei of quiescent human cells." Journal of Cell Science 112, no. 4 (February 15, 1999): 525–35. http://dx.doi.org/10.1242/jcs.112.4.525.

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The relative spatial positioning of chromosomes 7, 8, 16, X and Y was examined in nuclei of quiescent (noncycling) diploid and triploid human fibroblasts using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes and digital imaging. In quiescent diploid cells, interhomolog distances and chromosome homolog position maps revealed a nonrandom, preferential topology for chromosomes 7, 8 and 16, whereas chromosome X approximated a more random distribution. Variations in the orientation of nuclei on the culture substratum tended to hinder detection of an ordered chromosome topology at interphase by biasing homolog position maps towards random distributions. Using two chromosome X homologs as reference points in triploid cells (karyotype = 69, XXY), the intranuclear location of chromosome Y was found to be predictable within remarkably narrow spatial limits. Dual-FISH with various combinations of chromosome-specific DNA probes and contrasting fluorochromes was used to identify adjacent chromosomes in mitotic rosettes and test whether they are similarly positioned in interphase nuclei. From among the combinations tested, chromosomes 8 and 11 were found to be closely apposed in most mitotic rosettes and interphase nuclei. Overall, results suggest the existence of an ordered interphase chromosome topology in quiescent human cells in which at least some chromosome homologs exhibit a preferred relative intranuclear location that may correspond to the observed spatial order of chromosomes in rosettes of mitotic cells.

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Lenaz, Giorgio, Bruno Samori, Romana Fato, Maurizio Battino, Giovanna Parenti Castelli, and Ida Domini. "Localization and preferred orientations of ubiquinone homologs in model bilayers." Biochemistry and Cell Biology 70, no. 6 (June 1, 1992): 504–14. http://dx.doi.org/10.1139/o92-078.

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The localization of ubiquinone has been investigated in phospholipid bilayer vesicles in studies of fluorescence quenching of membrane-bound probes by ubiquinone homologs (Qn, where n is the number of the isoprenoid units of the chain). Fluorescence-quenching data obtained by using a set of anthroylstearate probes, having the fluorophore located at different depths, revealed that ubiquinone-3 is located throughout the whole bilayer thickness. From the bimolecular quenching constants in the membrane, lateral diffusion coefficients in two dimensions were calculated to span values of 10−7–10−6 cm2∙s−1. This suggests that ubiquinones laterally diffuse in a very fluid environment. On this basis, it is proposed that their translational diffusion in the bilayer takes place in two dimensions, with the quinone ring oscillating between the two bilayer surfaces within a hydrophobic environment not extending beyond the glycerol region. This model implies that the quinonic head is both settled near the polar surface of the bilayer and buried into the host hydrocarbon interior. This two-site distribution was confirmed for all Qn, except Q0, by their linear dichroism spectra in the bilayers provided by disc-like lyotropic nematic liquid crystals. These spectra also provided detailed information on the preferential orientations of the quinonic head of the different derivatives within the two sites. The mechanism by which the localization and orientation of Qn guest molecules inside the host bilayer is modulated by the isoprenoid chain length is discussed on a thermodynamical basis. Being that Qn is expected to be also widely contained in the highly curved cristae of the mitochondrial inner membrane, by using rod-like lyotropic nematic liquid crystals we searched out effects of the curvature of the host bilayer on those Qn distributions. The linear dichroism measurements reveal that Qn guest molecules are no longer obliged to find a partition between two different types of localizations when the host bilayer is highly curved. In this case all Qn, even the longest Q10, were found to stay parallel to the amphiphilic chains with a single site localization of the head near the polar interface. By the same linear dichroism technique, the local ordering of all Qn derivatives was also evaluated. The order parameters were found to be basically the same for all derivatives. This result is justified on the basis of the relaxation, caused by the surface curvature, of the lateral compression of the host chains.Key words: coenzyme Q (ubiquinone), fluorescence quenching, lateral diffusion, linear dichroism, model bilayers.

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Varshosaz, J., F. Hassanzadeh, H. Sadeghi Aliabadi, M. Nayebsadrian, M. Banitalebi, and M. Rostami. "Synthesis and Characterization of Folate-Targeted Dextran/Retinoic Acid Micelles for Doxorubicin Delivery in Acute Leukemia." BioMed Research International 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/525684.

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Folate and retinoic acid grafted/dextran (FA-RA/DEX) copolymers with different molecular weight of DEX were synthesized using carbonyldiimidazole and dimethylaminopyridine for targeted delivery of doxorubicin (DOX) in acute myelogenous leukemia (AML). The copolymers structure was confirmed by1H NMR and FTIR. Critical micelle concentration (CMC) of each copolymer was determined using pyrene as a fluorescent probe. DOX was loaded in micelles by the direct dissolution method. Physical properties of micelles, including particle size, zeta potential, drug loading efficiency, and drug release profiles, were examined. The orientation of the folate ligand on the surface of the micelles was studied by X-ray photoelectron spectroscopy (XPS) technique. The cytotoxicity of micelles loaded with DOX at different concentrations was studied in KG1 cells using MTT assay and their cellular uptake by flow cytometry technique. FTIR and1H NMR spectra confirmed successful production of the targeted micelles and XPS spectra showed the surface orientation of folate. R15D10F7copolymer produced micelles with particle size of 82.86 nm, polydispersity index of 0.3, zeta potential of −4.68 mV, drug loading efficiency of 96%, and release efficiency of 63%. DOX loaded in folate-targeted micelles of RA/DEX was more toxic than that in nontargeted micelles and free drug and seems promising in reducing drug resistance in AML.

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Karpicheva, Olga E., Armen O. Simonyan, Nikita A. Rysev, Charles S. Redwood, and Yurii S. Borovikov. "Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin." International Journal of Molecular Sciences 21, no. 20 (October 14, 2020): 7590. http://dx.doi.org/10.3390/ijms21207590.

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We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in β-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln147Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca2+.

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Schwarzacher, Trude, and J. S. Heslop-Harrison. "In situ hybridization to plant telomeres using synthetic oligomers." Genome 34, no. 3 (June 1, 1991): 317–23. http://dx.doi.org/10.1139/g91-052.

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The distribution of telomeric repeats in Hordeum vulgare (barley) and Secale cereale (rye) was studied by DNA–DNA in situ hybridization to root-tip chromosome preparations. Biotinylated synthetic oligomers, (TTTAGGG)6 and (CCCTAAA)6, homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat, were used as probes, and hybridization sites were detected by Texas red fluorescence or horseradish peroxidase catalyzed precipitation of diaminobenzidine. After examination in the light microscope, the same preparations were transferred to the electron microscope for high-resolution analysis. Sites of hybridization were visualized as single or double dots at the end of most chromosome arms. The sizes of the signal dots varied widely, indicating that individual telomeres may contain different numbers of repeats of the telomeric sequence. In contrast to many mammals, no telomere repeats were detected other than at the ends of the chromosomes. At interphase, signals were concentrated in one region of the nucleus, as would be expected from the Rabl orientation typical for dividing cells from cereal root tips.Key words: barley, rye, nuclear architecture, DNA–DNA in situ hybridization, telomeres.

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Matsuyama, Shutoku, Noriyo Nagata, Kazuya Shirato, Miyuki Kawase, Makoto Takeda, and Fumihiro Taguchi. "Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2." Journal of Virology 84, no. 24 (October 6, 2010): 12658–64. http://dx.doi.org/10.1128/jvi.01542-10.

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ABSTRACT The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.

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Paloncýová, Markéta, Marcel Ameloot, and Stefan Knippenberg. "Orientational distribution of DPH in lipid membranes: a comparison of molecular dynamics calculations and experimental time-resolved anisotropy experiments." Physical Chemistry Chemical Physics 21, no. 14 (2019): 7594–604. http://dx.doi.org/10.1039/c8cp07754a.

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The behavior of the fluorescent probe diphenylhexatriene (DPH) in different lipid phases is investigated. The rotational autocorrelation functions are calculated in order to model the time-resolved fluorescence anisotropy decay. The role of the order parameters is discussed.

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Emaminejad, Sam, Mehdi Javanmard, Chaitanya Gupta, Shuai Chang, Ronald W. Davis, and Roger T. Howe. "Tunable control of antibody immobilization using electric field." Proceedings of the National Academy of Sciences 112, no. 7 (February 3, 2015): 1995–99. http://dx.doi.org/10.1073/pnas.1424592112.

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The controlled immobilization of proteins on solid-state surfaces can play an important role in enhancing the sensitivity of both affinity-based biosensors and probe-free sensing platforms. Typical methods of controlling the orientation of probe proteins on a sensor surface involve surface chemistry-based techniques. Here, we present a method of tunably controlling the immobilization of proteins on a solid-state surface using electric field. We study the ability to orient molecules by immobilizing IgG molecules in microchannels while applying lateral fields. We use atomic force microscopy to both qualitatively and quantitatively study the orientation of antibodies on glass surfaces. We apply this ability for controlled orientation to enhance the performance of affinity-based assays. As a proof of concept, we use fluorescence detection to indirectly verify the modulation of the orientation of proteins bound to the surface. We studied the interaction of fluorescently tagged anti-IgG with surface immobilized IgG controlled by electric field. Our study demonstrates that the use of electric field can result in more than 100% enhancement in signal-to-noise ratio compared with normal physical adsorption.

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Davidsson, Åke, and Lennart B. Å. Johansson. "Electrochromism of fluorescent probes." Molecular Physics 57, no. 2 (February 10, 1986): 295–302. http://dx.doi.org/10.1080/00268978600100231.

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Kim, Jong Seung, and Duong Tuan Quang. "Calixarene-Derived Fluorescent Probes." Chemical Reviews 107, no. 9 (September 2007): 3780–99. http://dx.doi.org/10.1021/cr068046j.

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Diwu, Zhenjun, Cailan Zhang, Dieter H. Klaubert, and Richard P. Haugland. "Fluorescent molecular probes VI." Journal of Photochemistry and Photobiology A: Chemistry 131, no. 1-3 (February 2000): 95–100. http://dx.doi.org/10.1016/s1010-6030(99)00240-3.

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Zhou Yanyan, 周艳焰, 肖永川 Xiao Yongchuan, 孙力军 Sun Lijun, 陈阳 Chen Yang, 廖世彪 Liao Shibiao, 邱强 Qiu Qiang, 辜之木 Gu Zhimu, 戴能利 Dai Nengli, and 李进延 Li Jinyan. "Optical-Fiber Fluorescent Probes." Laser & Optoelectronics Progress 57, no. 1 (2020): 010003. http://dx.doi.org/10.3788/lop57.010003.

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Badugu, Ramachandram, Joseph R. Lakowicz, and Chris D. Geddes. "Cyanide-sensitive fluorescent probes." Dyes and Pigments 64, no. 1 (January 2005): 49–55. http://dx.doi.org/10.1016/j.dyepig.2004.04.002.

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Zhou, Jin, Paramesh Jangili, Subin Son, Myung Sun Ji, Miae Won, and Jong Seung Kim. "Fluorescent Diagnostic Probes: Fluorescent Diagnostic Probes in Neurodegenerative Diseases (Adv. Mater. 51/2020)." Advanced Materials 32, no. 51 (December 2020): 2070385. http://dx.doi.org/10.1002/adma.202070385.

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He, Juanjuan, Fanyong Yan, Depeng Kong, Qianghua Ye, Xuguang Zhou, and Li Chen. "Fluorescent Probes for Glutathione Detection." Current Organic Chemistry 20, no. 25 (September 27, 2016): 2718–34. http://dx.doi.org/10.2174/1385272820666160602125352.

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Hanson, George, and Bonnie Hanson. "Fluorescent Probes for Cellular Assays." Combinatorial Chemistry & High Throughput Screening 11, no. 7 (August 1, 2008): 505–13. http://dx.doi.org/10.2174/138620708785204090.

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Bruen, Danielle, Colm Delaney, Dermot Diamond, and Larisa Florea. "Fluorescent Probes for Sugar Detection." ACS Applied Materials & Interfaces 10, no. 44 (October 10, 2018): 38431–37. http://dx.doi.org/10.1021/acsami.8b13365.

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Al-Hassan, Khader A., and Wolfgang Rettig. "Free volume sensing fluorescent probes." Chemical Physics Letters 126, no. 3-4 (May 1986): 273–79. http://dx.doi.org/10.1016/s0009-2614(86)80082-3.

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Meyer, Andreas J., and Tobias P. Dick. "Fluorescent Protein-Based Redox Probes." Antioxidants & Redox Signaling 13, no. 5 (September 2010): 621–50. http://dx.doi.org/10.1089/ars.2009.2948.

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