Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy
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....
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2003-01-01
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| Series: | Journal of Pharmacological Sciences |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S134786131932571X |
| _version_ | 1818899775623266304 |
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| author | Hisayuki Ohata Hideyuki Yamada Takeharu Niioka Masayuki Yamamoto Kazutaka Momose |
| author_facet | Hisayuki Ohata Hideyuki Yamada Takeharu Niioka Masayuki Yamamoto Kazutaka Momose |
| author_sort | Hisayuki Ohata |
| collection | DOAJ |
| description | 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 Ca2+ 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-μm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 μm). When serial sectioning of 21 sequential images at 0.3-μm 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 Ca2+-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca2+]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 Ca2+ imaging in blood vessels in situ. |
| first_indexed | 2024-12-19T19:53:20Z |
| format | Article |
| id | doaj.art-ac42001aac284eb2a1cd74ff4b03a5a4 |
| institution | Directory Open Access Journal |
| issn | 1347-8613 |
| language | English |
| last_indexed | 2024-12-19T19:53:20Z |
| publishDate | 2003-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Pharmacological Sciences |
| spelling | doaj.art-ac42001aac284eb2a1cd74ff4b03a5a42022-12-21T20:07:54ZengElsevierJournal of Pharmacological Sciences1347-86132003-01-01933242247Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence MicroscopyHisayuki Ohata0Hideyuki Yamada1Takeharu Niioka2Masayuki Yamamoto3Kazutaka Momose4Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, JapanDepartment of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, JapanDepartment of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, JapanDepartment of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, JapanDepartment of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, JapanABSTRACT: 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 Ca2+ 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-μm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 μm). When serial sectioning of 21 sequential images at 0.3-μm 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 Ca2+-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca2+]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 Ca2+ imaging in blood vessels in situ.http://www.sciencedirect.com/science/article/pii/S134786131932571X |
| spellingShingle | Hisayuki Ohata Hideyuki Yamada Takeharu Niioka Masayuki Yamamoto Kazutaka Momose Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy Journal of Pharmacological Sciences |
| title | Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy |
| title_full | Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy |
| title_fullStr | Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy |
| title_full_unstemmed | Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy |
| title_short | Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy |
| title_sort | optical bioimaging from living tissue to a single molecule calcium imaging in blood vessel in situ employing two photon excitation fluorescence microscopy |
| url | http://www.sciencedirect.com/science/article/pii/S134786131932571X |
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