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Updated: Dec 24, 2025
A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Published on: March 30, 2020
A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Dinesh Jaishankar1, Cormac Cosgrove1, Ryan J Deaton2
1Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine, Northwestern University.
Insights
This study presents a rapid multispectral fluorescence imaging method for frozen tissues, enabling detection of up to six markers. This technique overcomes limitations of formalin-fixed paraffin-embedded (FFPE) tissues for marker detection.
Area of Science:
- Biomedical Imaging
- Histopathology
- Immunofluorescence
Background:
- Multispectral fluorescence imaging of formalin-fixed paraffin-embedded (FFPE) tissues allows multiplex marker detection.
- Limitations include restricted marker choice due to antibody availability and time-consuming staining protocols.
Purpose of the Study:
- To develop a rapid multispectral fluorescence imaging method for frozen tissues.
- To overcome FFPE limitations for marker detection and improve workflow efficiency.
Main Methods:
- Detailed protocol for staining mouse and human frozen tissues using specific fluorophore combinations.
- Utilized a semiautomated multispectral fluorescence imaging system for scanning, acquisition, and analysis.
- Employed machine learning software for cell phenotyping and quantitative analysis.
Main Results:
- Successfully stained and detected up to six different markers in a single frozen tissue section.
- Demonstrated the capability for quantitative analysis through machine learning-based cell phenotyping.
- The method is applicable to markers not detectable in FFPE tissues.
Conclusions:
- The described rapid method for frozen tissue multispectral fluorescence imaging offers a valuable alternative to FFPE-based approaches.
- It expands the range of detectable markers and streamlines the imaging workflow.
- This technique is particularly useful for markers with limited FFPE antibody availability.
Abstract:
Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.
