Total Internal Reflection Fluorescence Microscopy in Vascular Smooth Muscle

This chapter provides an overview of the basic concepts behind Total Internal Reflection Fluorescence Microscopy (TIRFM), and its application to the study of the function and regulation of plasmalemmal Ca²⁺-permeable channels in vascular smooth muscle. TIRFM utilizes an evanescent wave to selectively excite fluorophores in regions of a sample directly adjacent to the glass coverslip-buffer interface. The principles at the heart of TIRFM are based on the laws of refraction of light and properties of the refractive media. TIRFM relies on the ability to introduce light at angles exceeding a critical angle. A particular innovative use of TIRFM has been on the recording of Ca²⁺ signals produced by the opening of Ca²⁺-permeable channels at the plasma membrane of a cell, including vascular smooth muscle cells. The quality of vascular smooth muscle cells is critical for successful recording of sparklets using TIRFM. Single vascular smooth muscle cells can be obtained by enzymatic digestion of freshly dissected arteries from different vascular beds.