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Sequential, Multiplexed Immunofluorescent Imaging of Live Cells Based on DNA-Mediated Reversible Fluorophore
Li Xu1, Yuki Maeda1, Noriko Nakamura1,2,3
1Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Insights
This study introduces a novel sequential immunofluorescence imaging method for live cells. It uses DNA linkers for detachable fluorophores, enabling multiplexed protein analysis without harsh conditions.
Area of Science:
- Cell Biology
- Molecular Imaging
- Biochemistry
Background:
- Traditional immunofluorescence imaging is limited by spectral overlap, restricting simultaneous protein detection.
- Existing sequential imaging methods require harsh conditions, unsuitable for live-cell analysis.
- Live-cell protein dynamics and interactions remain challenging to study comprehensively.
Purpose of the Study:
- To develop a sequential, multiplexed immunofluorescence imaging method applicable to live cells.
- To enable repeated antibody-fluorophore attachment and detachment under mild physiological conditions.
- To overcome limitations of spectral overlap and harsh fixation in protein imaging.
Main Methods:
- Utilized DNA as a detachable linker between antibodies and fluorophores.
- Employed toehold-mediated strand displacement for controlled fluorophore binding and release.
- Applied the method to live A431 and A549 cell lines for multiplexed imaging.
Main Results:
- Successfully demonstrated at least six imaging cycles with three simultaneous fluorophores in live cells.
- Showcased the ability to image numerous protein markers within a single sample.
- Detected dynamic protein expression changes (EGFR, CD44, Integrin β1) during EGF stimulation over time.
Conclusions:
- The developed method allows for non-destructive, sequential multiplexed immunofluorescence imaging in live cells.
- This technique facilitates the comprehensive analysis of complex protein networks and their spatiotemporal regulation.
- Opens new avenues for studying dynamic cellular processes and protein interactions in real-time.
Abstract:
Given the spectral overlap of fluorophores, traditional immunofluorescence imaging is limited by the number of proteins that can be imaged simultaneously. Although sequential imaging techniques have been proposed, in which repeated staining and destaining are performed to obtain the merged image of several proteins, they are applied only to fixed cells presumably due to their harsh conditions. Therefore, observation and analysis of live cells have not been achieved with the sequential imaging approach. In this study, we develop a sequential, multiplexed immunofluorescence imaging method for live cells using DNA as a detachable linker to bind antibodies to fluorophores. The use of toehold-mediated strand displacement of DNAs enables the attachment and detachment of fluorophores under mild physiological conditions. Consequently, at least six imaging cycles and the simultaneous use of three different fluorophores are demonstrated in live A431 and A549 cells, indicating the potential of imaging numerous protein markers in a single sample. Furthermore, by performing sequential staining at different time points, the dynamic expression changes of multiple proteins (EGFR, CD44, and Integrin β1) during EGF stimulation can also be detected. This approach is expected to facilitate comprehensive analysis of complex protein networks and their spatiotemporal regulation in live cells.
