Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1407; p. 325
• Main Authors: Gerhardt, Matthias, Walz, Michael, Beta, Carsten
• Format: Journal Article
• Language: English
• Published: United States 01.01.2016
• Subjects: Actins - metabolism; Cell Membrane - metabolism; Cells, Cultured; Chemotaxis; Dictyostelium - physiology; Microscopy, Confocal - methods; Patch-Clamp Techniques; Dictyostelium discoideum; Patch clamp; Confocal microscopy; Chemotaxis; Actin dynamics; Excitable dynamics
• DOI: 10.1007/978-1-4939-3480-5_23

In this chapter, we describe how to shield a patch of a cell membrane against extracellularly applied chemoattractant stimuli. Classical patch clamp methodology is applied to allow for controlled shielding of a membrane patch by measuring the seal resistivity. In Dictyostelium cells, a seal resistivity of 50 MΩ proved to be tight enough to exclude molecules from diffusing into the shielded membrane region. This allowed for separating a shielded and a non-shielded region of a cell membrane to study the spatiotemporal dynamics of intracellular chemotactic signaling events at the interface between shielded and non-shielded areas. The spatiotemporal dynamics of signaling events in the membrane was read out by means of appropriate fluorescent markers using laser scanning confocal microscopy.