A Rapid Nanofocusing Method for a Deep-Sea Gene Sequencing Microscope Based on Critical Illumination

Area of Science:

• Biotechnology
• Microscopy
• Oceanography

Background:

• Deep-sea environments present significant spatial constraints for in-situ gene sequencing instrumentation.
• Optical imaging systems in deep-sea settings face defocusing issues due to temperature fluctuations and vibrations.
• Existing autofocusing methods may not meet the stringent requirements for deep-sea gene sequencing.

Purpose of the Study:

• To develop an edge detection algorithm for defocused images to enable precise autofocusing in deep-sea gene sequencing.
• To establish a nanometer-resolution defocus state detection model for compact microscopy applications.
• To address the limitations of volume, focusing accuracy, and speed in deep-sea optical imaging.

Main Methods:

• Proposed an edge detection algorithm for defocused images utilizing grayscale gradients.
• Developed a defocus state detection model leveraging the critical illumination light field for nanometer resolution.
• Integrated and tested the model on a prototype deep-sea gene sequencing microscope with a 20× objective.

Main Results:

• Achieved autofocusing within a ±40 μm dynamic range with 200 nm accuracy in a single iteration (160 ms).
• Refined focusing accuracy to 78 nm within a ±100 μm dynamic range over 1.2 seconds using multiple iterations and exposures.
• Demonstrated a compact, hardware-independent autofocusing solution suitable for deep-sea gene sequencing.

Conclusions:

• The proposed edge detection algorithm and defocus detection model effectively address autofocusing challenges in deep-sea gene sequencing.
• The system meets requirements for wide dynamic range, high speed, and accuracy without additional hardware.
• This technology offers a compact and efficient solution for in-situ deep-sea genetic analysis.