An open source 16-channel fluidics system for automating sequential fluorescent in situ hybridization (FISH)-based imaging

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荧光原位杂交（Fluorescent in situ hybridization, FISH）可获取固定细胞与组织内DNA/RNA靶标的空间信息。然而，采用多轮杂交流程的多重FISH成像技术，随着成像轮次增加，液体操作需求会导致实验工作量快速攀升。本文报道一款开源且低成本的流体控制系统，专为自动化连续FISH成像工作流量身定制。 该系统搭载流体模块，具备16个可寻址通道，流体采用正压驱动，并通过电磁阀实现通断控制，以此将FISH试剂输送至样本。系统还配备集成主控印刷电路板（Printed Circuit Board, PCB）的控制器，可支持最多120个电磁阀的控制，并允许用户通过串行通信对流体模块进行操控。 我们通过针对海拉（HeLa）细胞中的α卫星重复序列开展14轮连续杂交与成像实验，验证了本系统可实现自动化且稳定的流体更换流程。我们预期，这款简洁灵活的系统将为各类实验体系中开展多重原位检测的研究人员提供实用助力。 ### 附属文件清单 1. `bill_of_materials.xlsx`：该文件共包含8个工作表。除名为"Tubings"与"Remaining components"的工作表外，其余工作表均对应补充材料中分步搭建指南所描述的模块或组装套件的元器件清单。 2. `fluidics_system_controller_PCB_design_files.zip`：控制器印刷电路板（PCB）的KiCAD设计文件 3. `Eppendorf_tube_adaptor_3D_print_files.zip`：用于3D打印Eppendorf管适配器及用于排布固定适配器的槽式面板的STL文件 4. `laser_cut_acrylic_panel_organizer.ai`：用于激光切割组装结构框架及Eppendorf管适配器阵列面板的Illustrator文件 5. `controller_case_laser_cut.3dm`：用于激光切割控制器外壳的Rhinoceros建模设计文件 6. `case_for_pressure_regulator_laser_cut.pdf`：用于激光切割调压阀外壳的PDF文件 7. `large reservoir holder.stl`：用于3D打印大型储液罐支架的STL文件，该支架用于固定14轮连续FISH成像演示实验中清洗缓冲液与置换缓冲液所用的大型玻璃瓶 8. `user interface app for flow rate calibration.zip`：用于自动流量校准的用户界面应用的Python脚本文件 9. `NIS-Element Macro codes.zip`：包含NIS-Elements宏代码的文件夹，用于运行流体控制系统及14轮连续FISH成像演示实验中的流体更换流程 10. `Fluidics System Controller with ESP32`：包含控制器印刷电路板（PCB）固件源代码的文件夹 11. `image data`：包含图3、图4及图5图像数据的文件夹