The physiological properties of cells are typically investigated in ensembles yielding averaged data that mask heterogeneities present in any cell population. Single-cell analysis techniques based on microfluidic "lab on a chip" (LOC) devices have recently led to new insights into cellular physiology revealing the biological importance of cell variability. However, we are only at the beginning of understanding the complex nature of cellular constitution and function. Similarly is the study of individual cells in LOC devices a new field requiring further development. The aim of this thesis is to develop novel methods for functional and chemical single-cell analysis in controlled microfluidic environments using optical trapping for cell manipulation. Investigation of cellular function requires experimental techniques allowing us to monitor cell-to-cell variability of the response of a cell population to external stimuli and to sort out interesting cell phenotypes for further investigation. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions. Here, we developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Observed dose-dependent cytotoxic effects of the trapping laser on the cells' viability imposed an upper limit to the time periods during which optically trapped cells could be studied. For assays involving monitoring of cellular dynamics over several hours or days, we immobilized cell arrays formed by refractive optical tweezers in a hydrogel-matrix confined to a microfluidic chamber of 15 nL volume. We demonstrated multiplexing of this concept by integrating several chambers adjacent to each other in the same microfluidic device. In fluidic contact, each chamber could be exposed to a different reagent. Chemical single-cell analysis deals with identification and quantification of intracellular chemical species. Here, we report an alternative method for chemical cytometry using micro-beads functionalised with capture agents as intracellular affinity probe. Cell internalization of the beads followed by induced endosomal escape leads to binding of the target analyte to the surface of the beads. Using optical trapping with microfluidics, we demonstrate fast extraction of the beads from the cells and detection of bound target analyte under preservation of the single-cell analytics. In contrast to classical chemical cytometry, our method has the potential to reach high analytical sensitivity at simultaneously reasonable throughput.