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Immunofluorescence Microscopy

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

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Related Experiment Video

Updated: Dec 23, 2025

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Published on: March 30, 2020

Brightfield multiplex immunohistochemistry with multispectral imaging.

Larry E Morrison¹, Mark R Lefever¹, Lauren J Behman¹

¹Roche Tissue Diagnostics (Ventana Medical Systems, Inc.), 1910 E Innovation Park Drive, Tucson, AZ, 85755, USA.

Laboratory Investigation; a Journal of Technical Methods and Pathology

|April 29, 2020

Summary

This summary is machine-generated.

This study introduces a novel brightfield multiplex immunohistochemistry (IHC) method using new chromogens and multispectral imaging. This technique overcomes limitations of current methods, enabling faster and more detailed tumor microenvironment analysis for pathologists.

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Last Updated: Dec 23, 2025

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Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

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Area of Science:

Pathology
Biomedical Imaging
Biochemistry

Background:

Brightfield microscopy is standard for solid tumor diagnosis using hematoxylin and eosin (H&E) and immunohistochemistry (IHC).
Increasing understanding of tumor microenvironments necessitates multiplexed biomarker detection.
Current multiplex IHC relies on immunofluorescence, limited by brightfield chromogen spectral overlap and camera capabilities.

Purpose of the Study:

To overcome limitations in brightfield multiplex IHC by developing a novel approach using spectrally distinct chromogens and multispectral imaging.
To enable high-order multiplexed biomarker detection within the familiar brightfield microscopy framework.

Main Methods:

Developed new chromogens with narrow absorbance bands for multiplexed IHC.
Utilized matched illumination channels and monochrome imaging for spectral separation.
Implemented an automated rapid imaging system capturing multispectral data in under 1 second.
Validated assays on non-small cell lung cancer and prostate cancer tissue samples.

Main Results:

Successfully performed multiplex IHC with 4-5 chromogens plus hematoxylin.
Achieved spectral unmixing to resolve individual chromogens and remove crosstalk.
Demonstrated concordant biomarker expression with single stains in lung and prostate cancer models.
Automated system captured multispectral images rapidly.

Conclusions:

This novel brightfield multiplex IHC and multispectral imaging approach overcomes spectral limitations.
The technology combines high-order multiplexing with the speed and familiarity of brightfield microscopy.
Potential to accelerate the adoption of multiplexing in clinical pathology for enhanced tumor analysis.