Dual-modality imaging of immunofluorescence and imaging mass cytometry for whole-slide imaging and accurate segmentation

Abstract

Summary: Imaging mass cytometry (IMC) is a powerful technique capable of detecting over 30 markers on a single slide. It has been increasingly used for single-cell-based spatial phenotyping in a wide range of samples. However, it only acquires a rectangle field of view (FOV) with a relatively small size and low image resolution, which hinders downstream analysis. Here, we reported a highly practical dual-modality imaging method that combines high-resolution immunofluorescence (IF) and high-dimensional IMC on the same tissue slide. Our computational pipeline uses the whole-slide image (WSI) of IF as a spatial reference and integrates small-FOV IMC into a WSI of IMC. The high-resolution IF images enable accurate single-cell segmentation to extract robust high-dimensional IMC features for downstream analysis. We applied this method in esophageal adenocarcinoma of different stages, identified the single-cell pathology landscape via reconstruction of WSI IMC images, and demonstrated the advantage of the dual-modality imaging strategy. Motivation: Highly multiplexed tissue imaging allows visualization of the spatially resolved expression of multiple proteins at the single-cell level. Although imaging mass cytometry (IMC) using metal isotope-conjugated antibodies has a significant advantage of low background signal and little autofluorescence or batch effect, it has a low resolution that hampers accurate cell segmentation and results in inaccurate feature extraction. In addition, it is also challenging for the current IMC platform to acquire large cm2-sized regions, which limits its application and efficiency when studying larger clinical samples with non-rectangle shapes. To maximize the research output of IMC, we developed a dual-modality imaging method and proposed a comprehensive computational pipeline that combines IF and IMC. The proposed stitching algorithm in this study relies on the IF WSI as a reference for registering and stitching multiple IMC images obtained from different runs. Importantly, this algorithm is not reliant on the positional accuracy of the IMC imaging stage. Considering the possibility of slide removal and reinsertion during WSI IMC data acquisition, it becomes essential to align the IMC images with the comprehensive IF WSI that covers the entire tissue. This registration process plays a critical role in achieving precise spatial alignment, going beyond sole reliance on IMC coordinates.

Keywords

• CP: Imaging