A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

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.