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

Fast, high-throughput fluorescence imaging is essential for numerous biomedical applications, particularly in high-resolution volumetric tissue analysis.

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.

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 $\sim 16$ to $20\mu m$ thick mouse and human tissues prepared as frozen or formalin-fixed, paraffin-embedded sections.

Our approach achieves a 25-fold reduction in image acquisition time compared with conventional $z$ -scanning widefield microscopy. For example, a $2mm\times 2mm\times 16\mu m$ volume can be imaged in 4.7 min, down from $\sim 2h$ . We further demonstrate compatibility with multicolor imaging and successful application to nucleus segmentation for downstream analysis.

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.