A novel process for H&E, immunofluorescence, and imaging mass cytometry on a single slide with a concise analytics pipeline

Imaging mass cytometry (IMC) is a powerful spatial technology that utilizes cytometry time of flight to acquire multiplexed image datasets with up to 40 markers, via metal-tagged antibodies. Recent advances in IMC have led to the inclusion of RNAScope probes and multiple new analysis pipelines have led to faster analyses and better results. However, IMC still suffers from lower resolution (1 mu m(2) pixels) and relatively small regions of interest (ROIs) (< 2 mm(2)) compared to other, light-based microscope technologies. Capturing higher-resolution images on serial sections causes great difficulty when attempting to align cells and structures across serial sections, especially when observing smaller cell types and structures. Therefore, we demonstrate the combination of H&E and multiplex immunofluorescence imaging, for much higher resolution of the structural and cellular compartments found throughout the entire tissue section, with the high-dimensionality of IMC for specific ROIs on a single slide. Additionally, we demonstrate a simple and effective opensource cell segmentation and IMC analysis pipeline with previously published and freely available software.

Automated summary

Imaging mass cytometry (IMC) is a powerful spatial technology that allows for high-resolution imaging with multiple markers. However, it still faces challenges such as lower resolution and smaller regions of interest compared to other microscopy techniques. In order to overcome these limitations, the combination of H&E and multiplex immunofluorescence imaging is proposed to achieve higher resolution of structural and cellular compartments.