Abstract : In the recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical, easy to couple to commercial microscopes and provide powerful and stable light that can be triggered by TTL 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 localised illumination, typically achieved using lasers. Here we show that insertion of a long-pass (>411 nm) filter with 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 15-20 µm. We tested our system by performing calcium imaging experiments combined with L-glutamate or NMDA photo-release 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 budget to run similar experiments in a variety of physiological applications.
