[24] Confocal imaging analysis of intracellular ions in mixed cellular systems or in situ using two types of confocal microscopic systems

Ohata, Hisayuki; Yamamoto, Masayuki; Ujike, Yosuke; Rie, Gousei; Momose, Kazutaka

doi:10.1016/s0076-6879(99)07026-3

Cited by 4 publications

(6 citation statements)

References 19 publications

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“…1 and 2. We therefore performed simultaneous imaging of Ca 2+ spots in the apical and basal planes at a distance of 3 μm using our recently developed real‐time three‐dimensional confocal imaging system (Ohata et al . 1999) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Visualization of elementary mechanosensitive Ca²⁺‐influx events, Ca²⁺ spots, in bovine lens epithelial cells

Ohata

Tanaka

Maeyama

et al. 2001

The Journal of Physiology

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Section: Resultsmentioning

confidence: 99%

“…Real‐time three‐dimensional confocal imaging of [Ca 2+ ] i was performed as described previously (Ohata et al . 1999) using a real‐time confocal imaging system with synchronized high‐speed Z ‐axis scanning.…”

Section: Methodsmentioning

confidence: 99%

Visualization of elementary mechanosensitive Ca²⁺‐influx events, Ca²⁺ spots, in bovine lens epithelial cells

Ohata

Tanaka

Maeyama

et al. 2001

The Journal of Physiology

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“…In practice, we have established an imaging analysis protocol with respect to [Ca 2+ ] i in individual endothelial cells in situ with LSCM (12,13). However, LSCM led to increased photodamage with total time necessary to acquire images, especially during continuous imaging.…”

Section: +mentioning

confidence: 99%

“…When the extent of out of focus due to contraction or relaxation is too much to determine endothelial [Ca 2+ ] i , the xy-image was acquired as averaged image of several focal planes (approx. 20 mm) by xyz-scanning using a piezoelectric actuator controlled by a function generator (13). Z-axis resolution using TPEN can be controlled by this imaging technique.…”

Section: Photobleaching and Photodamage Of Tpemmentioning

confidence: 99%

Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy

et al. 2003

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Abstract. Recent developments in optoelectronics permit real-time Ca2+ imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca 2+ imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-mm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 mm). When serial sectioning of 21 sequential images at 0.3-mm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca ] i of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca 2+ imaging in blood vessels in situ.

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“…Our apparatus consisted of a multi-pinhole Nipkow disk-type confocal scanner (CSU10Z; Yokogawa Electric Co., Tokyo) and a high-speed cooled digital CCD camera fluorescence imaging system (ARGUS HiSCA; Hamamatsu Photonics, Hamamatsu), which permits acquisition of sequential fluorescence images at intervals of less than 20-ms (18). Neither addition of 3 mM LPA nor application of mechanical stress affected [Ca 2+ ]i as shown in Fig.…”

Section: Visualization Of Camentioning

confidence: 99%

Physiological and Pharmacological Role of Lysophosphatidic Acid as Modulator in Mechanotransduction

Ohata

Tanaka

Maeyama

et al. 2001

Japanese Journal of Pharmacology

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ABSTRACT-The mechanotransduction mechanism is believed to play an important role in maintenance of cellular homeostasis in a wide variety of cell types. In particular, the mechanotransduction system in vascular endothelial cells may be an essential mechanism for local hemodynamic control. Elevations in intracellular free Ca]i) are an important signal in the initial step of mechanotransduction and mechanosensitive (MS) cation channels are thought to be a putative pathway; however, the molecular mechanisms remain unclear. We found that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes the response of [Ca 2+ ]i to mechanical stress in several cell types. Employing real-time confocal microscopy, local increases in [Ca 2+ ]i in several regions within the cell during application of mechanical stress were clearly visualized in bovine lens epithelial and endothelial cells in the presence of LPA. The phenomenon was termed "Ca 2+ spots". Pharmacological studies revealed that Ca 2+ spots arise due to influx through MS channels. In this report, our data indicating the possible significance of LPA as an endogenous factor involved in regulation of mechanotransduction is reviewed. Furthermore, our findings suggest that the Ca 2+ spot is a novel phenomenon occurring as an elementary Ca 2+-influx event through MS channels directly coupled with the initial step in mechanotransduction.

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[24] Confocal imaging analysis of intracellular ions in mixed cellular systems or in situ using two types of confocal microscopic systems

Cited by 4 publications

References 19 publications

Visualization of elementary mechanosensitive Ca²⁺‐influx events, Ca²⁺ spots, in bovine lens epithelial cells

Visualization of elementary mechanosensitive Ca²⁺‐influx events, Ca²⁺ spots, in bovine lens epithelial cells

Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy

Physiological and Pharmacological Role of Lysophosphatidic Acid as Modulator in Mechanotransduction

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[24] Confocal imaging analysis of intracellular ions in mixed cellular systems or in situ using two types of confocal microscopic systems

Cited by 4 publications

References 19 publications

Visualization of elementary mechanosensitive Ca2+‐influx events, Ca2+ spots, in bovine lens epithelial cells

Visualization of elementary mechanosensitive Ca2+‐influx events, Ca2+ spots, in bovine lens epithelial cells

Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy

Physiological and Pharmacological Role of Lysophosphatidic Acid as Modulator in Mechanotransduction

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Visualization of elementary mechanosensitive Ca²⁺‐influx events, Ca²⁺ spots, in bovine lens epithelial cells

Visualization of elementary mechanosensitive Ca²⁺‐influx events, Ca²⁺ spots, in bovine lens epithelial cells