UV photolysis using a micromanipulated optical fiber to deliver UV energy directly to the sample

Abstract

UV photolysis of caged molecules is a powerful method for studying cellular signaling. However, UV energy is often delivered through the microscope objective which can make certain experiments difficult. We have evaluated the utility of delivering UV pulses directly to the sample through an optical fiber. Visible (635 nm) and UV (337 nm) lasers were coupled into a UV transmitting optical fiber which was micromanipulated over the sample under investigation. Positioning of the fiber, and thus the photolysis beam, was achieved using the visible laser which acted much like a flashlight. By controlling the size of the optical fiber it is also possible to control the area of the sample which is exposed to UV light. After positioning the fiber we demonstrate that the UV beam exiting the optical fiber reliably photolysed NP-EGTA that had been loaded into cells, resulting in an elevation of intracellular calcium. Additionally, caged norepinephrine in the bathing saline was photo-released to activate receptor-operated calcium signaling pathways. Since the delivery of the UV energy is independent of microscope configuration, this approach can be readily incorporated into wide-field fluorescence imaging, confocal microscopy and electrophysiological applications.