An open source 16-channel fluidics system for automating sequential fluorescent in situ hybridization (FISH)-based imaging
Description
Fluorescent in situ hybridization (FISH) can provide spatial information about DNA/RNA targets in fixed cells and tissues. However, the workflows of multiplexed FISH-based imaging that use sequential rounds of hybridization quickly become laborious as the number of rounds increases because of liquid handling demands. Here, we present an open-source and low-cost fluidics system that is purpose built for automating the workflows of sequential FISH-based imaging. Our system features a fluidics module with 16 addressable channels in which flow is positive pressure-driven and switched on/off by solenoid valves in order to transfer FISH reagents to the sample. Our system also includes a controller with a main printed circuit board that can control up to 120 solenoid valves and allows users to control the fluidics module via serial communication. We demonstrate the automatic and robust fluid exchange with this system by targeting the alpha satellite repeat in HeLa cell with 14 rounds of sequential hybridization and imaging. We anticipate that this simple and flexible system will be of utility to researchers performing multiplexed in situ assays in a range of experimental systems. bill_of_materials.xlsx: There are 8 sheets in this file. Except the sheets named ‘Tubings’ and ‘Remaining components’, each sheet lists components for a module and/or assembly described in the step-by-step building guide in the supplementary materials. fluidics_system_controller_PCB_design_files.zip: KiCAD design files for the controller PCB Eppendorf_tube_adaptor_3D_print_files.zip: STL files for 3D printing the Eppendorf tube adaptor and the slot panel that is designed for arranging and securing the adapters laser_cut_acrylic_panel_organizer.ai: Illustrator file for laser cutting the panels for assembling the structural frame and the Eppendorf tube adapter array controller_case_laser_cut.3dm: Rhrinoceros design file for laser cutting the controller case case_for_pressure_regulator_laser_cut.pdf: pdf file for laser cutting the pressure regulator case large reservoir holder.stl: STL file for 3D printing the large reservoir holders for securing large glass bottles for the washing buffer and displacement buffer used in the 14-round sequential FISH imaging demonstration experiment user interface app for flow rate calibration.zip: Python scripts for the user interface app for automatic flow rate calibration NIS-Element Macro codes.zip: Folder with NIS-Element Macro code for running the fluidics system and routines for the fluid exchange in the 14-round sequential FISH imaging demonstration experiment Fluidics System Controller with ESP32: Folder with source code for the firmware of the controller PCB image data: Folder with image data for Figure 3, 4, and 5