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Updated: Jun 5, 2026
Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells
Published on: June 13, 2018
Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Immune Structures in Human FFPE Tissue
Alex Y H Wong1,2, Yi Daniel Lu1,2, Ziyuan Zhao1,2,3
1Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA.
Insights
We developed volumetric cyclic immunofluorescence (v-CyCIF) to image 3D tissue structures in human samples. This method enables detailed study of immune cells and nerves, overcoming limitations of traditional 2D tissue analysis.
Area of Science:
- Immunology
- Neuroscience
- Biomedical Imaging
Background:
- Tissue-resident immune systems form complex 3D structures interacting with vasculature and nerves.
- Conventional 2D profiling struggles to analyze these interactions across multiple tissue sections.
- Volumetric imaging (LSFM) is established in animal models but limited in human FFPE tissues.
Purpose of the Study:
- To develop a method for multiplexed 3D imaging of immune cells and nerves in human specimens.
- To overcome limitations of analyzing complex neuroimmune interactions in formalin-fixed paraffin-embedded (FFPE) tissues.
- To enable multi-scale 3D profiling of clinical samples.
Main Methods:
- Development of a volumetric cyclic immunofluorescence (v-CyCIF) toolbox.
- Integration with a virtual Hematoxylin and Eosin (H&E) staining approach.
- Application to human specimens up to 1 mm thick, including re-embedding for high-resolution analysis.
Main Results:
- Successfully imaged neuroimmune interactions in normal and cancerous human tissues.
- Enabled immunoprofiling of intact secondary and tertiary lymphoid structures in 3D.
- Allowed high-plex, high-resolution analysis of subcellular structures and cell-cell interactions post-volumetric imaging.
Conclusions:
- v-CyCIF provides a flexible framework for multi-scale 3D profiling of clinical specimens.
- This approach overcomes key barriers to multiplexed imaging in human tissues.
- Enables advanced study of tissue architecture and cellular interactions in health and disease.
Abstract:
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.
