  We present a novel method for real-time monitoring and kinetic analysis of
fluorescence in situ hybridization (FISH). We implement the method using a
vertical microfluidic probe containing a microstructure designed for rapid
switching between a probe solution and a non-fluorescent imaging buffer. The
FISH signal is monitored in real time during the imaging buffer wash, during
which signal associated with unbound probes is removed. We provide a
theoretical description of the method as well as a demonstration of its
applicability using a model system of centromeric probes (Cen17). We
demonstrate the applicability of the method for the characterization of FISH
kinetics under conditions of varying probe concentration, destabilizing agent
(formamide) content, volume exclusion agent (dextran sulfate) content, and
ionic strength. We show that our method can be used to investigate the effect
of each of these variables and provide insight into processes affecting in situ
hybridization, facilitating the design of new assays.
