ArticleLight, science & applications2021
Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing.
Article in Light, science & applications, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed.
- Mesolens imaging in microbiology.Essays in biochemistry · 2026Review
- Single Fluorogens and Orientation-Localization Microscopy for Quantifying Chemical and Biomolecular Dynamics at the Nanoscale.Accounts of chemical research · 2026Article
- Deep unfolding blind source unmixing for multicolor fluorescence imaging.Biomedical optics express · 2026Article
- Enzymatic DNA Reaction Networks for Orchestrating Stimuli-Dependent Temporal Molecular Pulse.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Engineering quantum dot surfaces to preserve protein-DNA interactions for single-molecule visualization.Nano convergence · 2026Article
- Non-invasiveSurgery open science · 2026Article
- Chemical interactions in polyethylene glycol-induced condensates lead to an anomalous FRET response from a flexible linker-fluorescent protein crowding sensor.bioRxiv : the preprint server for biology · 2026Article
- Enhancing Biomarker Detection and Imaging Performance of Smartphone Fluorescence Microscopy Devices.Biosensors · 2025Article
- Imaging 3D cell cultures with optical microscopy.Nature methods · 2025Review
- Highly Efficient Calibration-Free Color Compensation Algorithm for Imaging Flow Cytometry.Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2025Article
- Fast segmentation and multiplexing imaging of organelles in live cells.Nature communications · 2025Article
- Creating coveted bioluminescence colors for simultaneous multi-color bioimaging.Science advances · 2025Article
- Droplet microfluidics for single-cell studies: a frontier in ecological understanding of microbiomes.FEMS microbiology reviews · 2025Review
- Electrochemical fluorescence modulation enables simultaneous multicolour imaging.Nature photonics · 2025Article
- Highly multiplexed fluorescence microscopy with spectrally tunable semiconducting polymer dots.Science advances · 2024Article
- Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism.Annual review of biophysics · 2024Review
- Hyperspectral imaging and dynamic region of interest tracking approaches to quantify localized cAMP signals.Biochemical Society transactions · 2024Review
- Article
- AutoUnmix: an autoencoder-based spectral unmixing method for multi-color fluorescence microscopy imaging.Biomedical optics express · 2023Article
- Single-Molecule Displacement Mapping Unveils Sign-Asymmetric Protein Charge Effects on Intraorganellar Diffusion.Nano letters · 2023Article
Corrections and comments
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Authors and funding
4 authors.
Funding
Abstract
The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.