Real-Time Monitoring of Fluorescence in situ Hybridization (FISH) Kinetics

TL;DR

This paper introduces a real-time monitoring technique for FISH that enables kinetic analysis and optimization of hybridization conditions using a microfluidic probe, improving assay design and understanding.

Contribution

The paper presents a novel microfluidic-based method for real-time FISH monitoring and kinetic analysis, including a theoretical framework and practical demonstration.

Findings

Effective real-time FISH monitoring demonstrated.

Insights into how probe concentration and chemical conditions affect hybridization.

Potential for optimizing FISH protocols and designing new assays.

Abstract

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.