| Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration. | |
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MedLine Citation:
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PMID: 19427524 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes. |
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Authors:
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Federico F Trigo; John E T Corrie; David Ogden |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2009-02-07 |
Journal Detail:
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Title: Journal of neuroscience methods Volume: 180 ISSN: 1872-678X ISO Abbreviation: J. Neurosci. Methods Publication Date: 2009 May |
Date Detail:
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Created Date: 2009-05-11 Completed Date: 2009-08-04 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 7905558 Medline TA: J Neurosci Methods Country: Netherlands |
Other Details:
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Languages: eng Pagination: 9-21 Citation Subset: IM |
Affiliation:
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Laboratoire de Physiologie Cérébrale CNRS UMR 8118, Université Paris Descartes, Paris 75006, France. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Acetic Acids
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chemistry Animals Calcium Signaling / physiology Calibration Cerebellum / physiology Electrophysiology / methods* Ethylenediamines / chemistry Glutamates / chemistry, radiation effects Indoles / chemistry, radiation effects Lasers / adverse effects* Light / adverse effects* Organ Culture Techniques Patch-Clamp Techniques / methods Photic Stimulation / adverse effects, methods Photochemistry / methods* Photolysis / radiation effects* Rats Rats, Sprague-Dawley Synaptic Transmission / physiology |
| Chemical | |
Reg. No./Substance:
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0/4-methoxy-7-nitroindolinyl-glutamate; 0/Acetic Acids; 0/Ethylenediamines; 0/Glutamates; 0/Indoles; 117367-86-9/1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis((oxycarbonyl)methyl)-1,2-ethanediamine |
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