研究目的
To address the limitations of current light sheet fluorescence microscopy (LSFM) related to throughput and sample handling by developing an automated high-throughput light sheet fluorescence microscope capable of rapid, three-dimensional imaging of live specimens with minimal manual intervention.
研究成果
The developed automated high-throughput light sheet fluorescence microscope significantly improves the throughput and simplicity of three-dimensional imaging of live specimens, demonstrating its utility by imaging fluorescent neutrophils in dozens of larval zebrafish. The instrument's design and capabilities suggest it could considerably expand the scope and impact of light sheet imaging in the life sciences.
研究不足
The design does not rotate the specimen about the travel axis, which could enhance image quality by allowing selection of particular orientations with minimal sheet distortion. The instrument's throughput is lower than plate-based instruments, and it does not integrate with commercial confocal microscopes.
1：Experimental Design and Method Selection
The instrument uses a fluidic system for specimen transport and positioning, integrated with light sheet excitation and detection optics. The design includes a horizontal fluidic path to prevent gravitational drift during imaging, with specimens automatically detected and positioned using brightfield microscopy and image-based registration.
2：Sample Selection and Data Sources
Larval zebrafish at 5 days post-fertilization (dpf) were used as model organisms, specifically those engineered to express green fluorescent protein (GFP) under the promoter myeloperoxidase for neutrophil imaging.
3：List of Experimental Equipment and Materials
Off-the-shelf parts were used for construction, with custom parts laser cut from acrylic sheets or 3D printed. Key components include solid-state lasers, an acousto-optic tunable filter (AOTF), a galvanometer mirror, objective lenses, a prism, a syringe pump, valves, and an sCMOS camera.
4：Experimental Procedures and Operational Workflow
Specimens are loaded into the tubing system manually, flowed through the system, automatically detected and positioned, imaged using LSFM, and then collected. The process includes brightfield imaging for positioning and fluorescence imaging for data collection.
5：Data Analysis Methods
Neutrophils were detected from image stacks using custom Python code involving coarse and fine thresholding, morphological operations, and object identification based on intensity and volume criteria.
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