Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Fast turnaround fabrication of silicon point-contact quantum-dot transistors using combined thermal scanning probe lithography and laser writing.

The fabrication of high-performance solid-state silicon quantum-devices requires high resolution patterning with minimal substrate damage. We have fabricated room temperature (RT) single-electron transistors (SETs) based on point-contact tunnel junctions using a hybrid lithography tool capable of both high resolution thermal scanning probe lithography and high throughput direct laser writing. The best focal z-position and the offset of the tip- and the laser-writing positions were determined in situ with the scanning probe. We demonstrate <100 nm precision in the registration between the high resolution and high throughput lithographies. The SET devices were fabricated on degenerately doped n-type >1020/cm3 silicon on insulator chips using a CMOS compatible geometric oxidation process. The characteristics of the three devices investigated were dominated by the presence of Si nanocrystals or phosphorous atoms embedded within the SiO2, forming quantum dots (QDs). The small size and strong localisation of electrons on the QDs facilitated SET operation even at RT. Temperature measurements showed that in the range 300 K > T > âˆ¼100 K, the current flow was thermally activated but at <100 K, it was dominated by tunnelling.