During the past 10 years, major developments in live-cell imaging methods have accompanied growing interest in the application of microfluidic techniques to biological imaging. The broad design possibilities of microfabrication and its relative ease of implementation have led to the development of a number of powerful imaging assays. Specifically, there has been great interest in the development of devices in which single cells can be followed in real-time over the course of several generations while the growth environment is changed. With standard perfusion chambers, the duration of a typical experiment is limited to one cell generation time. Using microfluidics, however, long-term imaging setups have been developed which can measure the effects of temporally controlled gene expression or pathway activation while tracking individual cells over the course of many generations. In this paper, we describe the details of fabricating such a microfluidic device for the purpose of long-term imaging of proliferating cells, the assembly of its individual components into a complete device, and then we give an example of how to use such a device to monitor real-time changes in gene expression in budding yeast. Our goal is to make this technique accessible to cell biology researchers without prior experience with microfluidic systems.