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多色虚拟荧光辐射差分显微成像

黄宇然 张智敏 董婉潔 徐良 韩于冰 郝翔 匡翠方 刘旭

黄宇然, 张智敏, 董婉潔, 徐良, 韩于冰, 郝翔, 匡翠方, 刘旭. 多色虚拟荧光辐射差分显微成像[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0080
引用本文: 黄宇然, 张智敏, 董婉潔, 徐良, 韩于冰, 郝翔, 匡翠方, 刘旭. 多色虚拟荧光辐射差分显微成像[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0080
HUANG Yu-ran, ZHANG Zhi-min, DONG Wan-jie, XU Liang, HAN Yu-bing, HAO Xiang, KUANG Cui-fang, LIU Xu. Multicolor virtual fluorescence emission difference microscopy[J]. Chinese Optics. doi: 10.37188/CO.2022-0080
Citation: HUANG Yu-ran, ZHANG Zhi-min, DONG Wan-jie, XU Liang, HAN Yu-bing, HAO Xiang, KUANG Cui-fang, LIU Xu. Multicolor virtual fluorescence emission difference microscopy[J]. Chinese Optics. doi: 10.37188/CO.2022-0080

多色虚拟荧光辐射差分显微成像

doi: 10.37188/CO.2022-0080
基金项目: 国家自然科学基金(No. 61827825、No. 62125504和No. 61735017); 浙江省自然科学基金重大项目(No. LD21F050002); 浙江省重点研究计划(No. 2020C01116); 之江实验室(No. 2020MC0AE01); 浙江省青年拔尖人才万人计划(No. 2020R52001); 中国博士后科学基金(No. BX2021272)
详细信息
    作者简介:

    黄宇然(1998—),男,陕西西安人,博士研究生,2020年于浙江大学获得学士学位,主要从事光学超分辨显微成像方面的研究。E-mail:huangyr@zju.edu.cn

    匡翠方(1977—),男,江西泰和人,博士,教授,博士生导师,主要从事光学超分辨显微成像方面的研究。E-mail:cfkuang@zju.edu.cn

  • 中图分类号: O436

Multicolor virtual fluorescence emission difference microscopy

Funds: Supported by National Natural Science Foundation of China (No. 61827825, No. 62125504 and No. 61735017); Major Program of the Natural Science Foundation of Zhejiang Province (No. LD21F050002); Key Research and Development Program of Zhejiang Province (No. 2020C01116); Zhejiang Lab (No. 2020MC0AE01); Zhejiang Provincial Ten Thousand Plan for Young Top Talents (No. 2020R52001); the China Postdoctoral Science Foundation (No. BX2021272).
More Information
  • 摘要: 荧光辐射差分显微成像是一种荧光染料普适性强、光毒性较低的超分辨成像技术。然而传统荧光辐射差分成像由于原理限制,其系统复杂度较高,稳定性低且成像速度受限。为了改进这些缺陷,本文设计搭建了一套多色虚拟荧光差分显微系统,并对该系统的成像方法和参数间的制约关系进行了分析,在已有的多色虚拟荧光辐射差分显微术原理的基础上,进一步考虑了信噪比和背景噪声等的影响,建立了可通过实验验证的虚拟荧光辐射差分显微成像模型。实验表明,本系统与方法具有结构简单、背景去噪能力强、荧光染料普适性强以及光毒性低等特性,成像分辨率较共聚焦提升了1.9倍,成像速度较传统的荧光辐射差分显微系统提升一倍,在三个波长上均获得了良好的成像效果,并在生物细胞成像中得到实验验证。

     

  • 图 1  虚拟荧光辐射差分显微系统简图

    Figure 1.  Sketch of virtual fluorescence emission difference microscopy

    图 2  系统成像模型,(a)中心通道、最左侧与最右侧通道的PSF的归一化强度分布;(b)光子重组后外轮廓匹配的PSF;(c)空心光斑图像仅来自中心通道时的虚拟实心PSF、空心PSF和vFED图像的PSF;(d)倾向于保证信噪比时的各图像PSF;(e)考虑背景噪声时的各图像PSF;(f)vFED与共聚焦以及实心光斑光子重组方法的成像性能对比

    Figure 2.  Imaging model of the system, (a) normalized intensity of PSFs in the center, leftmost and rightmost channels; (b) PSFs with matching outer contours after photon reassignment; (c) hollow PSF, and vFED PSF when hollow spot images from only the center channel virtual solid PSF; (d) PSF of each image when the signal-to-noise ratio tends to be guaranteed; (e) PSF of each image when the background noise is considered; (f) vFED, confocal, solid spot photon reassignment performance comparison

    图 3  荧光颗粒成像结果,(a)共聚焦图像;(b)实心光斑光子重组图像;(c)vFED图像,图(a−c)中方框内的区域高斯拟合得到的半高全宽分别为425.75 nm、320.96 nm、224.72 nm;(d)虚拟实心光斑图像;(e)空心光斑图像;(f)图(a−c) 中白色截线的归一化强度分布

    Figure 3.  Fluorescent particle imaging results, (a) confocal image; (b) solid spot photon reassignment image; (c) vFED image, the full width at half of the maximum obtained by Gaussian fitting of the regions in the box in (a−c). The widths are 425.75 nm, 320.96 nm and 224.72 nm, respectively,; (d) virtual solid spot image; (e) hollow spot image; (f) normalized intensity of the white truncation line in Figures (a−c)

    图 4  三色生物样品成像结果,(a)实心光斑光子重组图像;(b)(a)中方框内的区域的放大图;(c)同一区域的 vFED 图像

    Figure 4.  Imaging results of three-color biological samples, (a) solid spot photon reassignment image; (b) enlarged view of the area inside the box in (a); (c) vFED image of the same area

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  • 网络出版日期:  2022-06-16

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