Jun 29, 2026
3D Light-Sheet Fluorescence Imaging and Imaris-Based Analysis of Cleared Tissues
- Hanyu Liu1,
- Zhangfan Ding1,2,
- Anjali Kusumbe1
- 1Tissue and Tumor Microenvironments Lab, Cancer Discovery and Regenerative Medicine Program, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 636921, Singapore;
- 2State Key Laboratory of Oral Diseases, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
Protocol Citation: Hanyu Liu, Zhangfan Ding, Anjali Kusumbe 2026. 3D Light-Sheet Fluorescence Imaging and Imaris-Based Analysis of Cleared Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7qbw9lwz/v1
Manuscript citation:
Chen J, Ding Z, Biswas L, De Angelis J, Chatzis A, Kusumbe AP. Rapid 3D Immunolabeling and Light Sheet Microscopy for Quantitative Analysis of Intact Tissues. Comput Struct Biotechnol J. 2026 May 21;35(1):0121. doi: 10.34133/csbj.0121. PMID: 42179916; PMCID: PMC13191089.
Ding Z, Liu H, Chen J, Kusumbe AP. Protocol for ultrafast immunolabeling and 3D imaging of whole organs and large tissues. STAR Protoc. 2026 Jun 19;7(2):104623. doi: 10.1016/j.xpro.2026.104623. Epub 2026 Jun 6. PMID: 42247300; PMCID: PMC13260106.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 19, 2026
Last Modified: June 29, 2026
Protocol Integer ID: 319431
Keywords: 3D imaging, Imaris , Lightsheet microsope, sheet fluorescence microscope, sheet fluorescence microscopy, confocal microscopy, scanning confocal microscopy, optical tissue clearing with fluorescence, sheet fluorescence imaging, imaging of intact biological specimen, organ imaging, combining optical tissue clearing, dimensional architecture of tissue, resolution imaging, analysis of cleared tissue, dimensions with imaging depth, large volumetric dataset, imaging depth, toto imaging by lsfm, analysing large volumetric dataset, providing accurate volumetric dataset, fluorescence, 3d reconstruction, rapid image acquisition, 3d light, imaging, accurate volumetric dataset, imaging parameter, tissue structure, tissue clearing, intact biological specimen, tissue, toto imaging, cell, image acquisition, maintaining high spatial resolution, based 3d reconstruction, dimensional reconstruction, high spatial resolution, cleared tissue, surface rendering, spatial relationships between cell, intact tissue
Funders Acknowledgements:
Ministry of Education (MOE), Singapore: Academic Research Funds
Grant ID: #024983-00001
Ministry of Education (MOE), Singapore: Academic Research Funds
Grant ID: #025277-00026
European Research Council
Grant ID: StG: metaNiche, 805201
European Union’s Horizon 2020
Grant ID: no 857524
Disclaimer
The authors declare that they have no competing interests.
Abstract
Light-sheet fluorescence microscopy (LSFM) enables rapid three-dimensional (3D) imaging of intact biological specimens across micro- to mesoscale (μm–cm) dimensions with imaging depths of several millimeters1,3. Combining optical tissue clearing with fluorescence-labelled primary or secondary antibodies, LSFM provides cellular-resolution imaging while preserving the native three-dimensional architecture of tissues and organs2,3,4. Compared with conventional laser-scanning confocal microscopy, LSFM enables rapid image acquisition of large optically cleared specimens while maintaining high spatial resolution and minimizing photobleaching. Unlike serial section-based 3D reconstruction, in toto imaging by LSFM directly captures intact tissues, providing accurate volumetric datasets that preserve the spatial relationships between cells and tissue structures.
This protocol describes the operation of a light-sheet fluorescence microscope for whole-organ imaging, including sample mounting, image acquisition, and optimisation of imaging parameters. We further present a practical workflow for processing and analysing large volumetric datasets using Imaris software, including three-dimensional reconstruction, surface rendering, region-of-interest definition, visualisation, animation, and quantitative image analysis3,4.
Guidelines
A major advantage of 3D imaging is its ability to enable quantitative analysis of tissue architecture while preserving the native spatial relationships in the microenvironment1,2. The volumetric datasets generated by light-sheet fluorescence microscopy can be analysed using image software for three-dimensional visualisation, surface rendering, colocalisation, region-of-interest (ROI) analysis, animation, and quantitative measurements. This protocol presents an Imaris-based workflow for reconstructing, visualizing, and quantitatively analysing whole-organ datasets.
Materials
Reagents
Ethyl cinnamate (ECi; Sigma-Aldrich, cat. no. 112372)
Specific equipment
Miltenyi-LaVision Biotec UltraMicroscope II
LaVision BioTec ImSpector MACS
Software
• Imaris (version 9.9.0) (Bitplane, https://imaris.oxinst.com/packages)
• Imaris File converter (version 9.9.0) (Bitplane, https://imaris.oxinst.com/packages)
• Adobe Illustrator CC 2023 (Adobe, https://www.adobe.com/uk/products/illustrator)
• GraphPad Prism (version 9) (GraphPad, https://www.graphpad.com)
Troubleshooting
Problem
Air bubbles are trapped within the tissue during imaging.
Solution
Carefully inject ECi into the regions of the specimen using a fine needle without damaging the tissue. Removing trapped air bubbles minimises light refraction and improves image quality.
Problem
Imaris software is unavailable.
Solution
Alternative image analysis softwares to Imaris include, but are not limited to: Zen Black Zeiss, NIS Elements Nikon, MetaXpress Molecular Devices, Huygens Professional Scientific Volume Imaging, Imaris Bitplane, Acapella PerkinElmer, Gen5 BioTek, Cell Profiler Broad Institute, and ImgLib2.
Safety warnings
ECi is an irritant. Avoid direct contact with the skin, eyes, and mucous membranes. Handle with appropriate PPE.
Laser radiation can cause serious eye injury. Never look directly into the laser beam and always follow institutional laser safety procedures.
Ethics statement
Experiments involving animals were conducted in accordance with local ethical standards and obtained approval from Nanyang Technological University, Singapore
Before start
Before beginning this protocol, install the required software on a Windows 64-bit workstation. Image processing and analysis were performed using Imaris 10.1 (Oxford Instruments), which can be downloaded from the official Imaris website. Users are encouraged to complete the introductory tutorials before analysing experimental datasets to become familiar with image reconstruction, surface rendering, ROI definition, and quantitative analysis tools.
Imaris 10.1 tutorials:
We recommend a workstation equipped with a dedicated graphics processing unit (GPU), at least 64 GB RAM, and sufficient storage capacity for large volumetric datasets.
Lightsheet Fluorescent Microscope
Mount the samples and attach them to the sample holder using superglue.
Fill the tank with ECi (refractive index=1.57).
Safety information
Ethyl cinnamate (ECi) is an irritant. Avoid direct contact with the skin, eyes, and mucous membranes. Handle ECi in a chemical fume hood while wearing appropriate personal protective equipment (PPE).
In the maintenance section, inform the software about the objective you selected.
Place the sample holder on the platform. The sample is fixed on the holder with glue, and the holder is placed in the imaging chamber filled with ECi.
Position the sample in front of the objectives. The illumination objectives include LSFM 5X/0.1 foc and LSFM 10X/0.2 foc. The detection objectives include EC Plan-Neofluar 5X/0.16 foc (working distance =10.5 mm) and Clr Plan-Neofluar 20X/1.0 Corr (working distance =6.4 mm). Adjust illumination objective and detection objectives according to the refractive index of the imaging medium (Eci).
Adjust the power of the laser (10–20%), stepsize (2–4), thickness (3–6 μm), wavelengths (405, 488, 561, 639, and 785), and exposure time (10 μs-100 ms) according to the fluorescence intensity and imaging requirements to achieve the maximal signal and lowest background noise. The excitation and emission wavelengths of the dyes we used are listed below.
Note
| Dye | Excitation wavelength (nm) | Emission wavelength (nm) | |
| DAPI | 364 | 454 | |
| FITC | 492 | 520 | |
| CY3 | 550 | 570 | |
| CY5 | 648 | 662 |
Define the upper and lower sample stacks to determine the z-volume to be imaged, which depends on the thickness of the sample or the area of interest. For example, a kidney sample contains about 600–800 layers.
Start the acquisition and save the raw data.
Data analysis
Convert raw image data using the Imaris File Converter (Bitplane) and analyse the converted data using Imaris software (version 9.9.0, Bitplane).
Data processing
Utilize Imaris software to reconstruct and process the Z-sections of the light-sheet images, and define the Regions of Interest (ROI) for individual channels of certain tissue architectures, and then reconstruct the surface.
Basic analysis of whole organ
Set the resolution for different vessels or spots. Employ surface analysis XTensions tools within Imaris to perform vascular density quantification. Utilize the 3D crop tool to analyse the total tissue volume. Smooth the reconstructed surface and subtract the background using appropriate settings, and preview the final images before being generated.
Surface rendering of vasculature
Employ advanced tools of Imaris software to achieve animation displaying and quantitative analysis, facilitating comprehensive scientific insights.
Animation mode- video producing
Statistics mode- quantitative analysis of blood vessels and T cells
Conduct quantitative analysis based on the surface rendering and ROI definition. The open-source software, such as ImageJ, is able to perform basic data processing, but not advanced analyses, including surface and spot rendering and animation.
Protocol references
1. Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EH. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 2004 Aug 13;305(5686):1007-9. doi: 10.1126/science.1100035. PMID: 15310904.
2. Richardson DS, Lichtman JW. Clarifying Tissue Clearing. Cell. 2015 Jul 16;162(2):246-257. doi: 10.1016/j.cell.2015.06.067. PMID: 26186186; PMCID: PMC4537058.
3.Ding Z, Liu H, Chen J, Kusumbe AP. Protocol for ultrafast immunolabeling and 3D imaging of whole organs and large tissues. STAR Protoc. 2026 Jun 19;7(2):104623. doi: 10.1016/j.xpro.2026.104623. Epub 2026 Jun 6. PMID: 42247300; PMCID: PMC13260106.
4. Chen J, Ding Z, Biswas L, De Angelis J, Chatzis A, Kusumbe AP. Rapid 3D Immunolabeling and Light Sheet Microscopy for Quantitative Analysis of Intact Tissues. Comput Struct Biotechnol J. 2026 May 21;35(1):0121. doi: 10.34133/csbj.0121. PMID: 42179916; PMCID: PMC13191089.
Acknowledgements
A.P.K. is supported by the Ministry of Education (MOE), Singapore: Academic Research Funds (#024983-00001 and #025277-00026), the European Research Council (StG: metaNiche, 805201), and the European Union’s Horizon 2020 (no 857524).