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Updated: Mar 31, 2026
Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy
Published on: August 9, 2019
Optical clearing based cellular-level 3D visualization of intact lymph node cortex
Eunjoo Song1, Howon Seo1, Kibaek Choe1
1Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, South Korea.
Insights
Researchers developed an optical clearing method to visualize entire lymph node (LN) structures in 3D. This technique overcomes light scattering limitations, enabling deeper imaging and detailed analysis of immune cell organization and metastatic melanoma in LNs.
Area of Science:
- Immunology
- Biomedical Imaging
- Optical Engineering
Background:
- Lymph nodes (LNs) are crucial immune organs with complex 3D cellular and vascular structures.
- Conventional histology limits 3D analysis of LNs due to tissue disruption.
- Confocal microscopy offers 3D imaging but is limited by light scattering in biological tissues.
Purpose of the Study:
- To establish an optical clearing procedure for lymph nodes (LNs) to enable deep 3D imaging.
- To overcome the limitations of light scattering in intact LN tissue.
- To visualize cellular structures, vasculature, and metastatic cells within the entire LN cortex.
Main Methods:
- Developed and optimized an optical clearing protocol specifically for lymph node tissue.
- Utilized confocal microscopy for 3D volumetric imaging of cleared LN samples.
- Employed transgenic mice (H2B-GFP/actin-DsRed) and fluorescently labeled cells (B cells, T cells, melanoma) for visualization.
- Used fluorescently conjugated antibodies (anti-CD31, anti-LYVE-1) for vascular and lymphatic labeling.
Main Results:
- Achieved 3D volumetric visualization of the entire lymph node cortex, significantly improving imaging depth (over 4x).
- Successfully visualized key immune structures: T-cell zones, B-cell follicles, and germinal centers.
- Visualized metastatic melanoma cell colonies, vasculature, and lymphatic vessels within the LN cortex in 3D.
Conclusions:
- The established optical clearing method enables unprecedented deep 3D imaging of intact lymph nodes.
- This technique facilitates detailed analysis of immune microenvironments and disease processes, such as cancer metastasis.
- The method provides a powerful tool for studying LN structure-function relationships and immune responses.
Abstract:
Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellular-level structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex.
