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

Le-Mei Wang¹, Dhruvam Pandey¹, Wencai Zhang²

¹University of Central Florida, CREOL, The College of Optics and Photonics, Orlando, Florida, United States.

Journal of Biomedical Optics

|October 20, 2025

Summary

This summary is machine-generated.

This study introduces a novel microscopy technique combining multiplane and extended depth-of-field imaging for rapid, high-resolution volumetric tissue analysis. The new method significantly reduces imaging time for thick tissue sections, enabling faster biomedical research.

Keywords:

2.5D imaging extended depth of field high-throughput multiplane tissue imaging

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Area of Science:

Biomedical Imaging
Microscopy Techniques
Tissue Analysis

Background:

High-throughput fluorescence imaging is crucial for biomedical applications, especially high-resolution volumetric tissue analysis.
Current methods can be time-consuming, limiting the scope of tissue studies.

Purpose of the Study:

To develop and characterize an advanced imaging strategy for fast, high-resolution volumetric tissue analysis.
To combine multiplane microscopy with extended depth-of-field (EDOF) microscopy.

Main Methods:

Utilized 2.5D microscopy, an EDOF approach, integrated with a quad-plane image splitter.
Enabled simultaneous capture of four focal volumes for volumetric imaging of 16–20 μm thick mouse and human tissues.
Demonstrated compatibility with multicolor imaging and nucleus segmentation.

Main Results:

Achieved a 25-fold reduction in image acquisition time compared to conventional z-scanning widefield microscopy.
A 2 mm x 2 mm x 16 μm volume was imaged in 4.7 minutes, down from approximately 2 hours.
Successful application to nucleus segmentation for downstream analysis was demonstrated.

Conclusions:

The developed imaging technique offers a significant improvement in volumetric imaging speed for tissue analysis.
Provides a promising tool for researchers with minimal compromise in spatial resolution and sensitivity.
Facilitates faster and more efficient high-resolution volumetric tissue studies.