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 cells and solid tumor samples. Specifically, we have developed an ‘open-space’ μFISH implementation using a microfluidic scanning probe (MFP) technology. We used the MFP to shape nanoliter volumes of FISH probes hydrodynamically on cell monolayers and tissue sections to localized FISH reactions on selected cells with micrometer resolution. First, we demonstrated chromosomal enumeration in adherent cells using this method. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes, the rate-limiting step in conventional FISH. The continuous flow of probes on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes (CEP7 and CEP17). We further demonstrated spatially multiplexed μFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer. Further, we developed methods and protocols for implementing the rapidity of µFISH-based detection of cytogenetic biomarkers in formalin-fixed paraffin embedded (FFPE) tissue sections to direct the concept of micro-scale FISH towards a diagnostically relevant method. Using the MFP, we detected the human epidermal growth factor 2 (HER2) gene, an important breast cancer biomarker in tissue sections. We demonstrated the use of oligonucleotide FISH probes in ethylene carbonate-based buffer enabling rapid hybridization within < 1 min for chromosome enumeration and 10-15 min for assessment of the HER2 status in FFPE sections. We further demonstrated recycling of FISH probes for multiple sequential tests using a defined volume of probes by forming hierarchical hydrodynamic flow confinements and inverting the flows. The presented protocols and methods are compatible with the standard FISH protocols reduce the FISH probe consumption ~100-fold and the hybridization time 4-fold, resulting in an assay turnaround time of < 3 h, wheras a conventional FISH test in diagnostic laboratories takes > 24h. To investigate the biophysics of FISH assays, we further developed a novel method for real-time monitoring and kinetic analysis of FISH (RT-FISH). We designed a vertical microfluidic probe with microstructures designed for rapid switching between the probe solution for FISH probe incubation and a non-fluorescent imaging buffer for removing of the FISH hybridization mix from the cells. Individual FISH signals were monitored in real-time during the imaging buffer wash, while any signal associated with unbound probes were removed. We demonstrated the applicability of the method for characteriza-tion of FISH kinetics under conditions of varying probe concentration (Cen17), destabilizing agent (formamide) content, volume exclusion agent (dextran sulfate) content and ionic strength. This method can be used to investigate the effect of a multitude of variables affecting in situ hybridization. We believe the methods presented in this work may provide new insights and better understanding of in situ reaction processes and facilitate the design of new assays as well as more accurate theoretical or computational models describing in situ hybridization. A better understanding of the underlying biophysical mechanism of FISH could further enable transitioning from empirical towards rational design of FISH assays. Further, by overcoming some of the key limitations of the FISH assay, micro-scale FISH may become a powerful strategy to make FISH more pervasively and used routinely in diagnostic laboratories.