ORCID

0009-0004-2098-0357

Keywords

fluorescence microscopy; fast-volumetric imaging; extended depth of field; super-resolution; large FOV tissue imaging

Subject Categories

Bioimaging and Biomedical Optics | Biomedical Engineering and Bioengineering | Optics | Other Biomedical Engineering and Bioengineering

Abstract

Fluorescence microscopy is an indispensable tool in the biological sciences, enabling researchers to investigate intricate subcellular structures, particularly for volumetric studies. However, conventional optical microscopy for volumetric imaging remains fundamentally constrained by imaging speed and throughput. To bypass traditional serial z-scanning, we introduce an axially scan-free method using a phase layer cake to modulate the system's point spread function. This approach projects volumetric information onto a 2D plane in a single shot, offering high flexibility in tuning axial depth alongside simultaneous multicolor imaging with high spatial resolution and sensitivity. This dissertation divides these technical advancements into cellular and tissue imaging regimes. First, we report a 2.5D fluorescence microscopy (2.5DM) framework tailored for observing adherent cells. Combined with highly inclined illumination, it cuts acquisition time and reduces out-of-focus background by 2-fold compared to conventional epi-illumination. Instead of sequential z-scanning, our method uses a multi-layered glass for incoherent wavefront splitting, capturing a 3–4 μm thick sample in a single shot while maintaining high photon efficiency (95%) and high spatial resolution (< 300 - 400 nm). We successfully applied this framework to multicolor immunofluorescence imaging, volumetric super-resolution imaging, and single-particle tracking in living cells. Second, we present an extended 2.5DM design optimized for thicker, large-field-of-view tissue targets. This design captures four focal planes simultaneously on a single camera, enabling axially scan-free volumetric imaging of 16–20 μm thick frozen or formalin-fixed, paraffin-embedded (FFPE) mouse and human tissue sections. Our approach achieves a 25-fold reduction in acquisition time compared to widefield z-scanning microscopy; for instance, a 2 mm × 2 mm × 16 μm tissue volume is imaged in just 4.7 minutes instead of 2 hours. Altogether, this dissertation provides a reliable, photon-efficient approach to axially scan-free 3D microscopy, opening new pathways for next-generation volumetric imaging in biological research.

fluorescence microscopy; fast-volumetric imaging; extended depth of field; super-resolution; large FOV tissue imaging

Completion Date

2026

Semester

Summer

Committee Chair

Divliansky, Ivan/Wu, Shin-Tson/Zhang, Wencai

Degree

Doctor of Philosophy (Ph.D.)

College

College of Optics and Photonics

Department

College of Optics and Photonics

Format

PDF

Document Type

Dissertation

Language

English

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