Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium.

Kinosita, Kazuhiko; Itoh, Hiroyasu; Ishiwata, Shin’ichi; Hirano, Ken‐ichi; Nishizaka, Takayuki; Hayakawa, Tsuyoshi

doi:10.1083/jcb.115.1.67

Cited by 150 publications

(106 citation statements)

References 21 publications

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“…Collected fluorescence from the growth cones was focused on the cooled CCD camera (Micromax 512BFT; Roper Scientific, Duluth, GA). DIC images of the growth cones were recorded simultaneously with fluorescent images of the growth cones by using a homemade dual-view light-splitting system (Kinosita et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

Tani

Miyamoto²,

Fujimori³

et al. 2005

J. Neurosci.

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The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nM Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied Ͻ5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 m 2 s Ϫ1 . The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, ϳ4 m/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

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Section: Methodsmentioning

confidence: 99%

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

Tani

Miyamoto²,

Fujimori³

et al. 2005

J. Neurosci.

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“…A typical FA setup consists of linearly polarized excitation and two polarized detections of°uorescence, which are parallel or perpendicular to the direction of the polarized excitation. The parallel and perpendicular detection could be achieved sequentially by changing analyzers in the emission path or simultaneously by polarization beam splitter (PBS), 13 a Wollaston prism 17 or a Thompson prism. 39 The anisotropy and azimuth could be calculated through two relating pixels.…”

Section: Principle Of Fluorescence Polarization Microscopymentioning

confidence: 99%

“…It also provides a sca®old to organize the contents of cells in space and for intracellular transport with the motor proteins. Actin was¯rstly to be found to have an organized distribution of probed dipole orientations 13 and was imaged by various FPM techniques. [14][15][16][17][18] Myosin is an important motor protein moving along actin.…”

Section: Introductionmentioning

confidence: 99%

Super-resolution fluorescence polarization microscopy

Zhanghao

Gao

Jin³

et al. 2017

J. Innov. Opt. Health Sci.

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Fluorescence polarization is related to the dipole orientation of chromophores, making°uores-cence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. Several recent super resolution techniques have been applied to°uorescence polarization microscopy, achieving dipole measurement at nanoscale. In this review, we summarize both di®raction limited and super resolution°uorescence polarization microscopy techniques, as well as their applications in biological imaging.

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“…To simplify our task, we used a 'dual-view' emission path approach [156] in which the donor and acceptor signals are split using a dichroic mirror and bandpass filters but are imaged onto the same detector. Because one-half of the detector is used for each colour, only four excitation spots can be used.…”

Section: (I) Multipixel Hybrid Photodetectors Arraymentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

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Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

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Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium.

Abstract: . A new microscope technique, termed "W"

Cited by 150 publications

References 21 publications

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

Super-resolution fluorescence polarization microscopy

Development of new photon-counting detectors for single-molecule fluorescence microscopy

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