Dye Loading with Patch Pipettes

INTRODUCTION

This protocol describes the loading of individual cells with fluorescent probes via patch pipettes. The patch-clamp methodology has been successfully used for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A broad range of dyes can be used with this loading technique. Markers for morphological reconstruction (e.g., Lucifer yellow); ion-sensitive indicator dyes for monitoring second-messenger cascades (e.g., fura-2); and dye-labeled proteins for fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) studies are all suitable for patch-clamp loading. The most widespread application of this technique has been for Ca²⁺ imaging. Whole-cell patch-clamp recordings represent a versatile loading technique that allows combined electrophysiological and optical measurements at a quantitative level.