Microfluidics-based automatic immunofluorescence staining for single-molecule localization microscopy

Full automation of single-molecule localization microscopy (SMLM) is crucial for large-scale and high-throughput cellular imaging. It is well-known that SMLM typically consists of three major steps: immunofluorescence (IF) staining, optical imaging, and image processing. Currently, automation in optical imaging and image processing is almost complete; however, the automation of IF staining has been slow to advance, probably due to its complicated experimental operations. Here we present a low-cost automated method for IF staining, called super-resolution immunofluorescence staining by microfluidics (SRIF-fluidics). This method is suitable for both adherent and suspension cells and supports single-color and multi-color IF staining for SMLM. Our results show that SRIF-fluidics reduces antibody consumption by about 75% and shortens the sample preparation time from 5.6 hours (manual operation) to 2.5 ∼ 4.4 hours, depending on the sample types. Importantly, this method provides a satisfactory consistency of imaging results without sacrificing sample labeling quality. We believe that the method proposed in this paper is a necessary supplement to achieving fully automated SMLM and facilitating high-throughput SMLM in the near future.