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无标记高分辨率白光定量相位纳米显微镜系统

Label-free high-resolution white light quantitative phase nanoscopy system.

作者信息

Tayal Shilpa, Tiwari Shubham, Mehta Dalip Singh

机构信息

Bio-photonics and Green Photonics Laboratory, Department of Physics, Indian Institute of Technology Delhi, New Delhi, India.

出版信息

J Biophotonics. 2023 Apr;16(4):e202200298. doi: 10.1002/jbio.202200298. Epub 2023 Jan 15.

DOI:10.1002/jbio.202200298
PMID:36602467
Abstract

We present a high-resolution white light quantitative phase nanoscopy (WLQPN) system that can be utilized to visualize nanoparticles and subcellular features of the biological specimens. The five-phase shifting technique, along with deconvolution, is adopted to obtain super-resolution in phase imaging. The phase shifting technique can provide full detector resolution, making it beneficial as compared to the well-known Fourier analysis method. The Fourier transform method requires minimum angle of , where is maximum achievable spatial frequency. It limits the highest achievable resolution to much below the actual diffraction limit of the system. Thus, to obtain a high-resolution phase map of the biological specimen, a two-step process is adopted. First, the phase map is recovered using the five-phase shifting algorithm, with full detector spatial resolution. Second, the complex field is obtained from the recovered phase map and further processed using the Richardson Lucy total variation deconvolution algorithm to obtain super-resolution phase images. The present technique was tested on 1951 USAF resolution chart, 200 nm polystyrene beads and Escherichia coli bacteria using a 50×, 0.55NA objective lens. The 200 nm polystyrene beads are visually resolvable and subcellular features of the E. coli bacteria are also observed, suggesting a significant improvement in the resolution.

摘要

我们展示了一种高分辨率白光定量相纳米显微镜(WLQPN)系统,该系统可用于可视化生物样本中的纳米颗粒和亚细胞特征。采用五相移技术并结合去卷积,以在相成像中获得超分辨率。相移技术可以提供全探测器分辨率,与著名的傅里叶分析方法相比具有优势。傅里叶变换方法要求最小角度为 ,其中 是可实现的最大空间频率。这将可实现的最高分辨率限制在远低于系统实际衍射极限的水平。因此,为了获得生物样本的高分辨率相图,采用了两步法。首先,使用五相移算法恢复相图,并具有全探测器空间分辨率。其次从恢复的相图中获得复场,并使用理查森·露西总变分去卷积算法进一步处理,以获得超分辨率相图像。使用50×、0.55NA物镜在1951 USAF分辨率测试图、200 nm聚苯乙烯珠和大肠杆菌上对本技术进行了测试。200 nm聚苯乙烯珠在视觉上是可分辨的,并且也观察到了大肠杆菌的亚细胞特征,这表明分辨率有了显著提高。

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