Modular fluidic automation platform with integrated thermal control for multi-step molecular imaging workflows

Abstract

Automation of multi-step mRNA imaging protocols increases reproducibility and throughput in spatial biology, as many workflows require repeated buffer exchanges, precise timing, and controlled reaction conditions. Commercial automation platforms can be expensive, proprietary, and difficult to customise, limiting their use in most laboratories. Here, we present two open-source robots for the Rapid Amplified Multiplexed Fluorescent In-Situ Hybridization (RAM-FISH) workflow based on programmable delivery of fluids and integrated thermal control with no dedicated bubble trap requirement. The first robot is designed to perform the steps necessary for signal localization (Multiplexer), and the second performs signal removal (RemBot). Both robots function without manual supervision and conduct precise, repeatable buffer exchanges, temperature regulation, and timed reactions. Both can operate on free-floating and gel-embedded tissues and can be assembled using widely available components. The robots support iterative imaging workflows, enabling detection of multiple genes across sequential hybridization rounds within the same sample. By providing customizable and accessible robots, we lower the technical know-how barriers that need to be overcome to perform complex spatial imaging experiments and enable scalable, hands-free execution of multi-step multiplex-FISH.