Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Immune Structures in Human FFPE Tissue.

The tissue-resident immune system involves complex 3D assemblies that interact with extended structures such as blood vessels and nerves. These interactions are difficult to study using conventional 2D profiling because they span many tissue sections. In animal tissues, volumetric imaging approaches such as light-sheet fluorescence microscopy (LSFM) are widely used to study 3D tissue organization, with labelling often aided by genetically encoded reporters and vascular dyes. In contrast, LSFM of human specimens remains underdeveloped because most clinical samples are available only as formalin-fixed paraffin-embedded (FFPE) tissue, limiting labeling strategies primarily to dyes and antibodies. Here, we present a volumetric cyclic immunofluorescence (v-CyCIF) and virtual H&E toolbox that overcomes key barriers to multiplexed imaging of immune cells and nerves in human specimens up to 1 mm thick. We use v-CyCIF to study neuroimmune interactions in normal and cancer tissues and to immunoprofile intact secondary and tertiary lymphoid structures. Re-embedding and sectioning of specimens following volumetric imaging enables high-plex high-resolution analysis of subcellular structures and cell-cell interactions associated with immune cell activity. v-CyCIF therefore provides a flexible framework for multi-scale 3D profiling of clinical specimens across imaging formats and resolutions.