We present an experimental approach that allows exposure of cells plated on a single coverslip to multiple distinct environments. The original chamber design created a small region of injury using geometrically defined flows of the control and ischemic solutions. Modifications of the original chamber design presented in this article produce a range of flow patterns that can be advantageous for a variety of imaging applications. These applications include: experiments that address effects of different treatments applied to a cell network, parallel testing of negative and positive controls using a single coverslip, border effect studies, evaluation of the treatment's reversibility, and simultaneous monitoring of a cell layer loaded with different fluorescent indicators. The method also can be used to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in a normal or altered cardiac cell network. These possibilities are illustrated in cultures of neonatal rat cardiomyocytes using oxidant- and calcium-sensitive fluorescent indicators.