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

• Published in: Sensors (Basel, Switzerland) Vol. 24; no. 15; p. 5010
• Main Authors: Gao, Ming, Shu, Fengfeng, Zhou, Wenchao, Li, Huan, Wu, Yihui, Wang, Yue, Zhao, Shixun, Song, Zihan
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.08.2024
• Subjects: Accuracy; Algorithms; auto-focus; critical illumination; deep sea; edge gradient; gene sequencing; Genes; Imaging systems; Lasers; Lighting - instrumentation; Methods; Microscopy - instrumentation; Microscopy - methods; Salinity; Sequence Analysis, DNA - instrumentation; Sequence Analysis, DNA - methods; China; auto-focus; critical illumination; edge gradient; gene sequencing; deep sea
• ISSN: 1424-8220; 1424-8220
• DOI: 10.3390/s24155010

In the deep-sea environment, the volume available for an in-situ gene sequencer is severely limited. In addition, optical imaging systems are subject to real-time, large-scale defocusing problems caused by ambient temperature fluctuations and vibrational perturbations. To address these challenges, we propose an edge detection algorithm for defocused images based on grayscale gradients and establish a defocus state detection model with nanometer resolution capabilities by relying on the inherent critical illumination light field. The model has been applied to a prototype deep-sea gene sequencing microscope with a 20× objective. It has demonstrated the ability to focus within a dynamic range of ±40 μm with an accuracy of 200 nm by a single iteration within 160 ms. By increasing the number of iterations and exposures, the focusing accuracy can be refined to 78 nm within a dynamic range of ±100 μm within 1.2 s. Notably, unlike conventional photoelectric hill-climbing, this method requires no additional hardware and meets the wide dynamic range, speed, and high-accuracy autofocusing requirements of deep-sea gene sequencing in a compact form factor.