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 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.