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Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method
Jia-Ren Lin1, Mohammad Fallahi-Sichani2, Peter K Sorger1,2
1HMS LINCS Center &Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 USA.
Insights
Cyclic immunofluorescence (CycIF) enables high-content single-cell imaging by iteratively staining and bleaching fluorophores. This cost-effective, public-domain method enhances cellular analysis without specialized equipment.
Area of Science:
- Cellular and Molecular Imaging
- Immunology and Cell Biology
Background:
- Population-based studies often mask cellular heterogeneity.
- Mass cytometry (CyTOF) offers high multiplexing but lacks morphological data.
- Conventional immunofluorescence (IF) has limited multiplexing capabilities.
Purpose of the Study:
- To develop a high-multiplicity single-cell immunofluorescence method.
- To create a versatile and reproducible imaging technique.
- To enable advanced cellular analysis using standard laboratory equipment.
Main Methods:
- Developed cyclic immunofluorescence (CycIF), a novel imaging protocol.
- CycIF involves iterative cycles of four-color staining and fluorophore inactivation.
- The method utilizes standard reagents and instrumentation for multichannel image construction.
Main Results:
- Achieved high-multiplicity single-cell immunofluorescence.
- CycIF is a simple, versatile, and cost-effective procedure.
- The method is suitable for high-throughput assays and screening applications.
Conclusions:
- CycIF provides a public-domain solution for advanced single-cell imaging.
- This technique overcomes limitations of conventional IF and flow-based methods.
- CycIF facilitates detailed analysis of cellular physiology, morphology, and microenvironment.
Abstract:
Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.
