Research Papers: Imaging

Economic and simple system to combine single-spot photolysis and whole-field fluorescence imaging

[+] Author Affiliations
Nadia Jaafari, Marco Canepari

Inserm U836, Team 3, Grenoble Cedex 09, France

Université Joseph Fourier, Grenoble Institut des Neurosciences and Laboratoire Interdisciplinare de Physique (CNRS UMR 5588), France

Ion Channel Science and Therapeutics, Laboratories of Excellence

Mark Henson, Jeremy Graham

CAIRN Research Ltd., Faversham, UK

J. Biomed. Opt. 18(6), 060505 (Jun 13, 2013). doi:10.1117/1.JBO.18.6.060505
History: Received May 2, 2013; Revised May 16, 2013; Accepted May 16, 2013
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Abstract.  In recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical and easy to couple to commercial microscopes, and they provide powerful and stable light that can be triggered by transistor-transistor logic pulses in the range of tens of microseconds or shorter. LEDs are usually installed on the epifluorescence port of the microscope to obtain whole-field illumination, which is ideal for fluorescence imaging. In contrast, photolysis or channelrhodopsin stimulation often requires localized illumination, typically achieved using lasers. Here we show that insertion of a long-pass (>411nm) filter with an appropriately sized pinhole in the epifluorescence pathway, combined with dual UV/visible illumination, can produce efficient whole-field visible illumination and spot UV illumination of 15 to 20 μm. We tested our system by performing calcium imaging experiments combined with L-glutamate or N-methyl-D-aspartic acid (NMDA) photorelease in hippocampal neurons from brain slices or dissociated cultures, demonstrating the ability to obtain local activation of NMDA receptors exclusively in the illuminated spot. The very inexpensive and simple system that we report here will allow many laboratories with limited budgets to run similar experiments in a variety of physiological applications.

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© 2013 Society of Photo-Optical Instrumentation Engineers

Citation

Nadia Jaafari ; Mark Henson ; Jeremy Graham and Marco Canepari
"Economic and simple system to combine single-spot photolysis and whole-field fluorescence imaging", J. Biomed. Opt. 18(6), 060505 (Jun 13, 2013). ; http://dx.doi.org/10.1117/1.JBO.18.6.060505


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