Scattering Reduction and Axial Resolution Enhancement in Light-Sheet Fluorescence Microscopy

IF 2 3区 物理与天体物理 Q3 BIOCHEMICAL RESEARCH METHODS
Yang Zhang, Runze Li, Xianghua Yu, Hao Miao, Ruiwen Yang, Xing Li, Junwei Min, Yanlong Yang, Dan Dan, Taiqiang Dai, Liang Kong, Baoli Yao
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引用次数: 0

Abstract

Light-sheet fluorescence microscopy (LSFM) provides an ideal tool for long-term observation of live specimens due to its low photodamage and fast volumetric imaging speed. The wavefront distortions in the illumination path of LSFM will reduce the intensity and broaden the light-sheet thickness, thereby degrading the image quality. We propose to use the wavefront shaping technique to reduce the scattering effect and shrink the light-sheet thickness. Scanning the refocused laser beam to generate LS improves both the fluorescence intensity and the axial resolution. The axial resolution can be further enhanced by subtracting the two images captured via double scanning the samples with the refocused beam and the uncorrected scattered beam for each slice. The axial resolution is improved from 2.2 ± 0.3 to 1.5 ± 0.2 μm across the field of view of 270 μm × 270 μm. The effectiveness of the wavefront shaping subtraction method is demonstrated by imaging fluorescent beads and Aspergillus conidiophores behind a scattering medium.

Abstract Image

光片荧光显微镜散射减少和轴向分辨率增强。
光片荧光显微镜(LSFM)由于其低光损伤和快速的体积成像速度,为长期观察活标本提供了理想的工具。在LSFM的照明路径中,波前畸变会降低光强,使光片厚度变宽,从而降低图像质量。我们建议采用波前整形技术来减小散射效应和缩小光片厚度。扫描重聚焦激光束产生LS,提高了荧光强度和轴向分辨率。通过对每个切片用重新聚焦的光束和未校正的散射光束进行双重扫描捕获的两幅图像进行减去,可以进一步提高轴向分辨率。在270 μm × 270 μm视场范围内,轴向分辨率从2.2±0.3 μm提高到1.5±0.2 μm。波前整形减法的有效性证明了成像荧光珠和分生孢子曲霉背后的散射介质。
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来源期刊
Journal of Biophotonics
Journal of Biophotonics 生物-生化研究方法
CiteScore
5.70
自引率
7.10%
发文量
248
审稿时长
1 months
期刊介绍: The first international journal dedicated to publishing reviews and original articles from this exciting field, the Journal of Biophotonics covers the broad range of research on interactions between light and biological material. The journal offers a platform where the physicist communicates with the biologist and where the clinical practitioner learns about the latest tools for the diagnosis of diseases. As such, the journal is highly interdisciplinary, publishing cutting edge research in the fields of life sciences, medicine, physics, chemistry, and engineering. The coverage extends from fundamental research to specific developments, while also including the latest applications.
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