Document Detail

Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration.
MedLine Citation:
PMID:  19427524     Owner:  NLM     Status:  MEDLINE    
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.
Federico F Trigo; John E T Corrie; David Ogden
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-02-07
Journal Detail:
Title:  Journal of neuroscience methods     Volume:  180     ISSN:  1872-678X     ISO Abbreviation:  J. Neurosci. Methods     Publication Date:  2009 May 
Date Detail:
Created Date:  2009-05-11     Completed Date:  2009-08-04     Revised Date:  2014-02-19    
Medline Journal Info:
Nlm Unique ID:  7905558     Medline TA:  J Neurosci Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  9-21     Citation Subset:  IM    
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MeSH Terms
Acetates / chemistry
Calcium Signaling / physiology
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, Sprague-Dawley
Synaptic Transmission / physiology
Grant Support
MC_U117532185//Medical Research Council
Reg. No./Substance:
0/4-methoxy-7-nitroindolinyl-glutamate; 0/Acetates; 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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