Symmetric Off‐Axis Spectral Encoding Enables High‐Throughput Six‐Color Optical Sectioning Imaging

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 mm 2 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.