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Cell volume is a critical parameter for the biology of living species and has an impact on virtually all cellular functions. In Schizosaccharomyces pombe, the regulation of cell size has been the focus of intense investigation, and mechanisms that couple size control with cell cycle progression have been identified. In fission yeast, cell length at division is generally used as a proxy for determining cell size. However, it only allows for an inaccurate evaluation of this critical parameter and neglects potential changes in cell morphology and diameter, which can strongly impact cell volume. Until recently, one of the major obstacles for studying the complexity of cell size regulation in fission yeast has been the lack of a robust method for high-throughput, direct measurement of single-cell volume. Here, we provide a comprehensive protocol for S. pombe cell volume determination based on the Fluorescence eXclusion method and microfluidics technologies. This approach makes it possible to reliably describe cell volume and its distribution in populations of fission yeast cells., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

By implementing an external feedback loop one can tightly control the expression of a gene over many cell generations with quantitative accuracy. Controlling precisely the level of a protein of interest will be useful to probe quantitatively the dynamical properties of cellular processes and to drive complex, synthetically-engineered networks. In this chapter we describe a platform for real-time closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmo-stress responsive promoter and playing with the osmolarity of the cells environment, we demonstrate that long-term control can indeed be achieved for both time-constant and time-varying target profiles, at the population level, and even at the single-cell level.

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.