A recent study in Nature Photonics presents a new multicolor fluorescence imaging method that uses electrochemical fluorescence modulation to visualize multiple cellular targets with overlapping emission spectra.
The technique enhances standard fluorescence microscopes by using changes in electrochemical potential to create distinct fluorescence responses, improving both specificity and sensitivity without needing hardware changes.
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Advancements in Fluorescence Imaging
Fluorescence microscopy is widely used in biology to study cell structures and activity with high detail. Traditional multicolor imaging depends on fluorophores with separate emission spectra and requires filters and beamsplitters. This limits the number of fluorophores that can be used and demands careful selection.
While methods like spectral unmixing and fluorescence lifetime imaging help, they often rely on complex equipment. There’s still a need for simpler and more flexible ways to do multicolor imaging.
How the Method Works
The researchers developed a practical way to use electrochemical fluorescence modulation with regular fluorescence microscopes. They used an indium tin oxide (ITO)-coated glass coverslip, which acts as both the imaging surface and the working electrode.
By connecting the ITO to a potentiostat, they could control the electrochemical potential during imaging. This allowed them to modulate the fluorescence intensity of different dyes and create unique electrochemical spectra (EC spectra) for each one.
To test the method, they labeled microtubules and nuclei with fluorophores such as ATTO 655 and STAR RED. By scanning the electrochemical potential over time, they recorded time-lapse images showing each dye’s unique modulation behavior.
They then used least-squares fitting with non-negativity constraints to separate the overlapping signals from mixed samples. This enabled accurate identification of each fluorophore using only a single-color optical setup.
The method worked not only with standard fluorescence imaging but also with super-resolution stimulated emission depletion (STED) microscopy. Using a redox system (cysteamine and ferricyanide) in a low-oxygen buffer, they achieved effective full-cell modulation. This showed the method can enhance multicolor imaging without changes to existing microscope hardware.
Key Findings: Expanding Imaging Possibilities
The results showed that electrochemical fluorescence modulation could reliably separate signals from multiple fluorophores, even when their emission spectra overlapped. Using distinct EC spectra of dyes like ATTO 655 and STAR RED, the researchers were able to unmix signals from cellular structures labeled with overlapping fluorophores.
They used least-squares fitting to accurately separate these signals, achieving six-color imaging across just three spectral channels on a standard confocal microscope. The unmixing process consistently kept signal overlap (cross-talk) to a minimum.
The method also worked well for resolving four fluorophores within a single spectral channel, even in heavily labeled samples. It was compatible with both widefield and super-resolution STED microscopy. In STED, the team achieved high-resolution four-color imaging using only one pair of excitation and depletion lasers, while also keeping photobleaching and spectral interference low.
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Potential Uses in Research
This technique could be useful in many areas, including cell biology, neuroscience, and biomedical imaging. It offers a straightforward and cost-effective way to do multicolor imaging with better accuracy. Since it works with existing microscopes, it avoids the need for advanced optics or major system upgrades.
By allowing detailed imaging of dynamic cellular processes and protein interactions, the method supports studies of complex biological systems. It also holds promise for high-throughput imaging tasks and could be adapted for diagnostic or therapeutic research.
Overall, it’s a practical tool for expanding multicolor imaging capabilities with greater precision and flexibility.
Journal Reference
Yang, Y., et al. (2025). Electrochemical fluorescence modulation enables simultaneous multicolour imaging. Nat. Photon. DOI: 10.1038/s41566-025-01672-7, https://www.nature.com/articles/s41566-025-01672-7
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