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

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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.

Introduction

A significant tool for cell biology is the ability to uncage bio-active molecules over a rapid time-scale, while observing the consequences for cellular signaling. With the development of several caged compounds including caged transmitters such as glutamate and norepinephrine, caged intracellular messengers such as IP3, cAMP and calcium, as well as other molecules including ATP, there is a body of growing literature in which uncaging has increased our understanding of cellular signaling (Mulkey and Zucker, 1993, Neher and Zucker, 1993, Tse et al., 1993, Zucker, 1993, Heinemann et al., 1994, Katz and Dalva, 1994, Kamiya and Zucker, 1994, Makings and Tsien, 1994, Wang and Augustine, 1995, Morgan and Jacob, 1996, Neveu and Zucker, 1996, Otis et al., 1996).

To uncage bio-active molecules, a UV source of energy is required. This can take several forms. For example, flashlamps have been successfully employed as has epi-illumination from a mercury arc lamp. Alternatively, argon-ion lasers coupled with fast mechanical shutters have proven highly successful. Despite the utility of this approach, high costs of certain lasers, difficulty of experimental operation, and the need to simultaneously image with wide-field illumination while providing focal uncaging, have all hampered the use of this method in cell biology. We have used a different approach, in which we deliver UV pulses to the sample through an optical fiber while imaging intracellular calcium levels with wide-field fluorescence microscopy. We find this method is simple to use and works reliably on cultured cells. Furthermore, there are several advantageous features to this approach in that the success of the procedure is not limited by the objective in use, photolysis is not limited by the type of microscope employed, and when simultaneous full-field imaging is performed, photolysis does not interfere with the excitation optical pathway.

Section snippets

Culture preparation

Primary cultures of mixed hippocampal neurons and astrocytes from 0- to 2-day-old postnatal rats were prepared by a modification of previously described procedures (Basarsky et al., 1994, Trudeau et al., 1996). Neurons and astrocytes were plated onto poly-l-lysine- (1 mg/ml, MW 100 000; Sigma, St. Loius, MO) coated glass coverslips and used in experiments after 9–14 days. Culture medium was supplemented with 5% fetal calf serum (Gibco BRL, Grand Island, NY) and Mito+serum additives

Results and discussion

To initially test whether our photolysis unit delivers sufficient energy to uncage molecules we used caged fluorescein. The fiber was immersed into a fura-2 solution, which fluoresces when excited in near UV, to confirm the delivery of pulsed light to the tip of the fiber (Fig. 2A). Once we had confirmed light delivery, we thoroughly washed away the fura-2 solution and caged fluorescein was introduced to the bath. This caged fluorophore is non-fluorescent until photolysed (Fig. 2B). Application

Acknowledgments

The authors would like to thank Dr Barbara Innocenti for helpful comments on the manuscript, and Robert Doyle for providing neuronal cultures used in some of these studies. This work was supported by grants from the NIH (NS24233 and NS37585) to PGH, the Iowa State University Biotechnology Council (to PGH and VP), and from the Whitehall Foundation (VP). The authors would like to thank Prairie Technologies (Waunakee, WI) for a gift of UV transmitting optical fiber and for proprietary knowledge

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