Multiplane 2.5D microscopy for high-throughput high-resolution tissue imaging.

Significance: Fast, high-throughput fluorescence imaging is essential for numerous biomedical applications, particularly in high-resolution volumetric tissue analysis. Aim: We aim to develop an imaging strategy that combines the strengths of multiplane microscopy and extended depth-of-field (EDOF) microscopy and to characterize its performance on tissue samples. Approach: We employed 2.5D microscopy, an EDOF approach optimized for high-resolution imaging, and integrated it with a quad-plane image splitter. This technique enables simultaneous capture of four focal volumes using a single camera, allowing volumetric imaging of ∼ 16 to 20    μ m thick mouse and human tissues prepared as frozen or formalin-fixed, paraffin-embedded sections. Results: Our approach achieves a 25-fold reduction in image acquisition time compared with conventional z -scanning widefield microscopy. For example, a 2    m m × 2    m m × 16    μ m volume can be imaged in 4.7 min, down from ∼ 2    h . We further demonstrate compatibility with multicolor imaging and successful application to nucleus segmentation for downstream analysis. Conclusions: This imaging technique provides a promising tool for tissue analysis, offering significant improvements in volumetric imaging speed with minimal compromise in spatial resolution and sensitivity.