Symmetric Off-Axis Spectral Encoding Enables High-Throughput Six-Color Optical Sectioning Imaging.
- 1MoE Key Laboratory for Biomedical Photonics Wuhan National Laboratory for Optoelectronics Huazhong University of Science and Technology Wuhan China.
Abstract
Multicolor fluorescence microscopy is central to biological imaging, but simultaneous detection of additional colors typically reduces throughput, increases spectral crosstalk, or lowers signal-to-noise ratio. We developed symmetric off-axis spectral encoding microscopy (sym-OSEM), which uses paired spatial encoding and differential decoding to separate fluorescence channels and suppress common-mode out-of-focus background. The differential decoding requires no specimen-specific reference spectrum, point spread function, or training data. All fluorophores are excited and detected simultaneously, so acquisition time is largely independent of channel count within the demonstrated two- to six-color configurations. Simulations and experiments using cell preparations, a serially imaged 300 μm-thick mouse-brain volume, and lymph node sections validated the approach. Four-color imaging of U2OS cells covered 6.362 mm2 min-1, 59-fold faster than a commercial confocal microscope under the tested acquisition settings, while retaining comparable image quality. Simultaneous six-color imaging resolved the distributions of immune-cell and stromal markers in lymph-node tissue. Sym-OSEM therefore provides a strategy for combining multicolor separation, optical sectioning, and high-throughput acquisition in a single imaging framework.
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BibTeX
@article{zhao2026symmetric,
title = {{Symmetric Off-Axis Spectral Encoding Enables High-Throughput Six-Color Optical Sectioning Imaging.}},
author = {Jiangjiang Zhao and Zhangheng Ding and Zhan Zhang and Jie Zhang and Shuhong Qi and Jing Yuan},
journal = {Nanophotonics (Berlin, Germany)},
year = {2026},
month = nov,
doi = {10.1002/nap2.70367},
url = {https://europepmc.org/article/MED/42999791},
note = {CC BY},
}
RIS
TY - JOUR
TI - Symmetric Off-Axis Spectral Encoding Enables High-Throughput Six-Color Optical Sectioning Imaging.
AU - Jiangjiang Zhao
AU - Zhangheng Ding
AU - Zhan Zhang
AU - Jie Zhang
AU - Shuhong Qi
AU - Jing Yuan
PY - 2026
DA - 2026/11/07
JO - Nanophotonics (Berlin, Germany)
DO - 10.1002/nap2.70367
UR - https://europepmc.org/article/MED/42999791
AB - Multicolor fluorescence microscopy is central to biological imaging, but simultaneous detection of additional colors typically reduces throughput, increases spectral crosstalk, or lowers signal-to-noise ratio. We developed symmetric off-axis spectral encoding microscopy (sym-OSEM), which uses paired spatial encoding and differential decoding to separate fluorescence channels and suppress common-mode out-of-focus background. The differential decoding requires no specimen-specific reference spectrum, point spread function, or training data. All fluorophores are excited and detected simultaneously, so acquisition time is largely independent of channel count within the demonstrated two- to six-color configurations. Simulations and experiments using cell preparations, a serially imaged 300 μm-thick mouse-brain volume, and lymph node sections validated the approach. Four-color imaging of U2OS cells covered 6.362 mm2 min-1, 59-fold faster than a commercial confocal microscope under the tested acquisition settings, while retaining comparable image quality. Simultaneous six-color imaging resolved the distributions of immune-cell and stromal markers in lymph-node tissue. Sym-OSEM therefore provides a strategy for combining multicolor separation, optical sectioning, and high-throughput acquisition in a single imaging framework.
SN - 2192-8606
SN - 2192-8614
ER -