Micro-scale fluorescence in situ hybridization

In this thesis, we aimed to leverage some of the unique hydrodynamic properties of fluidics to make fluorescence in situ hybridization (FISH) assays more efficient, and to elucidate the underlying physical mechanisms surrounding this important class of cytogenetic techniques. Hydrodynamic phenomena are ubiquitous in living organisms and can be used to manipulate cells or emulate physiological microenvironments experienced in vivo. Hydrodynamic effects influence multiple cellular properties and processes, including cell morphology, intracellular processes, cell-cell signalling cascades and reaction kinetics, and play an important role at the single-cell, multicellular and organ level. Selected hydrodynamic effects can also be leveraged to control mechanical stresses, analyte transport as well as local temperature within cellular microenvironments. With a better understanding of fluid mechanics at the micrometer length scale and the advent of microfluidic technologies, a new generation of microfluidic tools that provides control over cellular microenvironments or emulates physiological conditions with exquisite accuracy for research and diagnostics is now emerging. FISH is a powerful cytogenetic technique and is used in research and diagnostics to detect cytosolic and nuclear nucleic acid targets at the single molecule level. Although FISH is a very specific technique it is not regularly used in diagnostics due to long assay times, expensive reagents (FISH probes) and the lack of trained personnel in diagnostic laboratories. To make FISH more pervasively used, the microfluidic community has developed tools and methods - μFISH implementations- using ‘closed’ devices to solve some bottlenecks of FISH assays. These devices however are in direct physical contact with the cytological sample or require cell immobilization within the microchannels, which is challenging. To tackle the problems of closed systems, in this thesis we designed and developed a non-contact microfluidics-based FISH implementation for analysis of ...