ArticleScientific reports2022
Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation.
Article in Scientific reports, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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17 citing papers in PubMed, 34 citations in OpenAlex.
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- STED Super-Resolution Microscopy for Studying Actin Cytoskeleton Organization.Methods in molecular biology (Clifton, N.J.) · 2026Article
- A fluorescence lifetime separation approach for FLIM live-cell imaging.Journal of microscopy · 2026Article
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- Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes.Life science alliance · 2024Article
- Enhanced optical imaging and fluorescent labeling for visualizing drug molecules within living organisms.Acta pharmaceutica Sinica. B · 2024Review
- Protein nanobarcodes enable single-step multiplexed fluorescence imaging.PLoS biology · 2023Article
- Spatial resolution enhancement in photon-starved STED imaging using deep learning-based fluorescence lifetime analysis.Nanoscale · 2023Article
- Identifying STEDable BFMolecules (Basel, Switzerland) · 2023Article
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.
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