Visualizing Cell Type–Specific Glycan Profiles in Tissue Sections With Glycan Painting

Background

Hematoxylin and eosin (H&E) staining is one of the most fundamental and widely used staining methods and has served as a cornerstone of pathology, histology, and developmental biology for over 100 years [1]. By providing color contrast in otherwise colorless and transparent tissue sections, H&E staining enables visualization of overall tissue architecture and cellular morphology and has long been employed as a fundamental method for histological observation. In H&E staining, the basic dye hematoxylin stains cell nuclei containing RNA and DNA as blue-violet, whereas the acidic dye eosin stains the cytoplasm and extracellular matrix as pink-red. Together, these contrasting dyes reveal a wide range of morphological features, including cell size, nuclear shape, cellular density, and tissue architecture, allowing cell types to be inferred through comprehensive visual assessment.

However, because the identification of cell types in H&E staining relies primarily on morphological features, the ability to distinguish morphologically similar cell populations remains difficult. In addition, H&E staining does not provide direct information regarding cellular function or the state of molecular expression. To address these limitations, immunofluorescent staining using antibodies against cell type–specific proteins has become widely adopted [2]. Immunofluorescent staining enables the visualization of cell type–specific molecular markers, providing information at the molecular expression level that complements conventional morphological observations. While immunofluorescent staining is useful, this staining method also has several limitations, including a restricted multiplex staining capacity, the need to optimize staining conditions, and dependence on predefined molecular markers.

The development of a technology capable of identifying various cell types in biological tissues as distinct color patterns would have broad significance across research fields such as cell and developmental biology, neuroscience, and pathology. In particular, the intuitive visualization of spatial cellular organization and intracellular relationships may provide new perspectives on tissue structure beyond conventional morphology-based analysis. Here, the focus is placed on cell-specific profiles of glycans, which are widely distributed on the cell surface, within cells, and in the extracellular matrix, where they play crucial roles in development and tissue organization [3]. Furthermore, a vast number of glycan-binding molecules, collectively known as lectins, exist throughout the biological world [4]. However, individual glycans do not exhibit strict cell-type specificity, and lectins possess relatively broad and overlapping glycan-binding specificities, limiting their utility as cell identification markers in a conventional single-probe staining approach. In contrast, combinations of glycan profiles exhibit cell-type specificity [5].

We therefore developed a novel staining method termed glycan painting in which differences in glycan profiles are translated into distinct color patterns, enabling cell populations to be distinguished based on their glycan composition [6]. In brief, merged images generated from triplex fluorescence staining of tissue sections using different lectins labeled with distinct fluorescent dyes allow different cell populations to be visualized as characteristic colors. In this report, ConA, WGA, and LEL were selected because they recognize distinct glycan structures and provide distinct staining patterns, enabling diverse RGB color combinations for visual discrimination of different tissue and cell populations. This approach is based on the additive mixing of three primary colors of light (red, green, and blue) and is analogous to the color-generation mechanism of an LCD monitor. In this study, glycan painting was applied to comprehensively visualize mouse embryonic tissues by leveraging the broad distribution of glycans and the overlapping glycan-binding specificity of lectins, features traditionally regarded as limitations, to generate full-color tissue images. Further optimization of lectin combinations may improve the performance of glycan painting, depending on the tissue type and biological application.