Microfluidic tools for single cell analysis

Industrial biotechnology plays an important role in the production of fine chemicals, pharmaceuticals and biofuels. Microorganisms are used to convert sustainable resources, e.g. sugars, into high value products. Yet, it has emerged that clonal populations can differ phenotypically. The cause of this heterogeneity is manifold and has been related to microgradients in large scaled bioreactors and the stochasticity in gene expression as well as metabolic pathways. Single cell analysis focuses on unravelling the underlying mechanisms of population based heterogeneity. Here, conventional methods such as flow cytometry and high resolution time-lapse microscopy have been applied intensively for many years. Yet, these methods do not offer the temporal and spatial resolution needed for the investigation the complex processes in cells. Microfluidics has become a promising new tool for studying bacteria populations at the single cell level with reduced reagents consumption and stable environmental conditions through fast exchange of heat and mass. For achieving a better understanding of the underlying processes in microbial population heterogeneity, new tools were integrated into microfluidic devices, such as cell trapping and cultivation as well as the sampling of single cells. In order to study solely single bacteria cells with a high temporal and spatial resolution, a new microfluidic device was established as described in Publication I, allowing the immobilization of single Escherichia coli cells in micron scaled barrier structures. Hence, cell growth can be monitored without the interference of descending cells, which are constantly removed by the media flow holding the cell inside the trap. High-throughput analysis of small bacterial microcolonies of up to 500 cells was realized as shown in Publication II by integrating hundreds of shallow growth chambers into microfluidic channels, restricting the growth of single cells to a monolayer. Mass-transport inside monolayer growth chambers solely relies on diffusion reducing shear forces to a minimum. Hence, growth of single cells in monolayer growth chambers can be followed at a high-resolution. Furthermore, by using automated image analysis cell lineages can be reconstructed and analyzed in high-throughput manner. A novel loading procedure was developed, to guarantee isogenic (clonal) starting conditions in microfluidic monolayer growth chambers - one bacteria cell per chamber (Publication III). Here, an artificial introduced air bubble is applied to temporally distort the flow profile, leading to convective flow inside the monolayer growth chambers. Subsequently, single cells get stuck inside the shallow chambers. After the air bubble is removed mass-transport inside the growth chambers is solely dominated by diffusion again. In this study, the process of air bubble based cell loading as well as loading efficiency was characterized and optimal growth condition after cell loading could been shown. High-throughput cultivation and analysis of small microbial colonies in monolayer growth chambers not allowed for retrieving single cells of interest for further analysis on or outside the microfluidic device. Publication IV introduced a new concept by integrating high-throughput microfluidic single-cell analysis and optical tweezers for sampling single cells from small microcolonies. Optical tweezers applies a highly focused infrared laser beam in order to trap and manipulate micron sized objects and cells with the force of light only. It was shown that single cells of Escherichia coli could be dragged in and out of shallow growth pockets. Characterization of the possible influences due to infrared laser irradiation were carried out and obtained result showed that short exposure times