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Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice (usually a few hundred nanometers to a few micrometers) of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction (e.g. using a cylindrical lens). A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because LSFM scans samples by using a plane of light instead of a point (as in confocal microscopy), it can acquire images at speeds 100 to 1000 times faster than those offered by point-scanning methods. Comparison of different microscopy illumination modalities (LSFM: lightsheet fluorescence microscopy, WF: widefield microscopy, CF: confocal microscopy). LSFM combines good z-sectioning (as confocal) and only illuminates the observed plane. This method is used in cell biology and for microscopy of intact, often chemically cleared, organs, embryos, and organisms. Link https://en.wikipedia.org/wiki/Light_sheet_fluorescence_microscopy 


Automated cell tracking in the Zebrafish Digital Embryo The images show a montage of DSLM microscopy data (right half of embryo: animal view, maximum-intensity projection) and the Digital Embryo (left half of embryo) with color-encoded cell migration directions. Color code: dorsal migration (cyan), ventral migration (green), toward or away from body axis (red or yellow), toward yolk (pink). Reference: Keller et al. 2008, Science.


Dual-view iSPIM improves axial resolution in 4D embryonic a b diSPIM SDCM imaging. (a) Selected diSPIM maximum-intensity projections of
GFP-labeled histones in nematode embryo, from 786 time-point volumetric series. Projections were computed 60 degrees relative to y axis.
(b) Comparison between diSPIM (left column), single view iSPIM (middle) and SDCM (right) at the same time point in embryogenesis. Lower two rows: higher magnification views of boxed nuclei in top two rows. All times are hours:minutes post fertilization. Projections are taken at
0 or 90 degrees relative to y axis. Reference: Wu et. al. 2013 Nature Biotechnology



The Idea: OpenSPIM is an Open Access platform for applying and enhancing Selective Plane Illumination Microscopy (SPIM). We hope that OpenSPIM in its radical openness will demonstrate that the benefits brought to science by the Open Source approach apply equally well to hardware. SPIM principle: The SPIM technology offers fast, optically-sectioning, minimally-invasive 3D acquisition of fluorescing specimen over time. It achieves that by focusing a thin laser light-sheet into the specimen, taking two-dimensional images of the illuminated slice with a perpendicularly positioned detector (CCD camera). Three-dimensional stacks are obtained by moving the specimen orthogonal to the light-sheet between consecutive images. By mounting the sample in a rigid medium, e.g. agarose, and hanging it into the sample chamber in front of the detection lens, it is possible to rotate the sample and collect 3d stacks from multiple angles (views). Reference: https://openspim.org


2014年诺贝尔化学奖获得者Eric Betzig在获奖之后不久,于2014年10月在Science期刊上又发表了一项重量级技术”Lattice light-sheet microscopy”。在接受华盛顿邮报采访时,Betzig教授表示这项技术对于生命科学研究的影响甚至将高于他获诺贝尔奖的超分辨技术(“this development will have more of an impact on biological research than the work that earned him a Nobel Prize”)。我们来了解下什么是Lattice light-sheet microscopy,暂且先翻译为晶格光片显微术。介绍开始之前,同学们需要对light-sheet microscopy(光片显微术)有个简要的认识,与传统荧光显微镜或共聚焦显微镜不同,光片显微镜的照明光轴和检测光轴是垂直的,即下右图橘色light-sheet与物镜光轴是垂直的。而传统荧光显微镜,照明和检测光轴是平行的,成180度,见下图。


为什么我们需要这样特殊的光学架构呢?其一是,这样的光毒性比较小,只有显微成像的样品区域是被激发光照射的,而传统荧光显微镜在焦面以外的样品区域都会被照射激发;其二是,提高Z轴分辨率,因为包括共聚焦技术在内Z轴分辨率一直是受限的。在光片显微镜之前,全内反射荧光显微镜(TIRF)可以将Z轴分辨率提高至100-200nm,但TIRF只能观察靠近容器玻璃片表面的样品。光片显微镜的观察区域比较灵活。比较直观的认识,如果需要进一步提高Z轴分辨率,那么照明光片本身的厚度需要越小越好。这项技术发展的过程中,科学家们在这方面做了很多成功的尝试,从最初用柱面镜(Cylindrical lens)产生光片(SPIM),到后来通过振镜扫描聚焦激光光斑实现更窄的光片(DSLM),以及双光子光斑扫描。前面提到的这几种方法都是用高斯光束(Gaussian beam)来实现的,后来物理学家们发现,用贝塞尔光束(Bessel beam)可以实现更细更窄的光片,来提高Z轴分辨率。上右图,从左至右分别是SPIM, DSLM, 2-photon SPIM, Bessel beam的示意图。

但贝塞尔光束并不是最理想的,尽管它在焦面上确实可以很窄,但是同时会产生非焦平面的激发光。Betzig教授结合了结构光照明(structure illumination microscopy, SIM)的方法,抵消了贝塞尔光束这些负影响,同时也实现了超分辨成像。为了减少成像时间,他们把贝塞尔光束分成七部分,同时扫描,而这样正好形成一个Lattice(晶格)的结构。就这样,Lattice light-sheet把三种领先的技术(即light-sheet, bessel beam, 还有超分辨SIM)结合在一起,实现了活体系统里更高3D光学分辨率、更小光毒性的显微成像技术。



一般来说,Lattice light sheet microscopy系统主要特点如下:
1) 理想样品中,XYZ三维分辨率可实现230 x 230 x 370 nm
2) 照明光片的厚度可以小于600 nm,极大减少光漂白和光毒性
3) 活体样品中可以实现高速成像,300帧/秒
4) 在斑马鱼或果蝇等胚胎中,可实现成像深度50 um

The Idea: OpenSPIM is an Open Access platform for applying and enhancing Selective Plane Illumination Microscopy (SPIM). We hope that OpenSPIM in its radical openness will demonstrate that the benefits brought to science by the Open Source approach apply equally well to hardware. SPIM principle: The SPIM technology offers fast, optically-sectioning, minimally-invasive 3D acquisition of fluorescing specimen over time. It achieves that by focusing a thin laser light-sheet into the specimen, taking two-dimensional images of the illuminated slice with a perpendicularly positioned detector (CCD camera). Three-dimensional stacks are obtained by moving the specimen orthogonal to the light-sheet between consecutive images. By mounting the sample in a rigid medium, e.g. agarose, and hanging it into the sample chamber in front of the detection lens, it is possible to rotate the sample and collect 3d stacks from multiple angles (views). Reference: https://openspim.org


2014年诺贝尔化学奖获得者Eric Betzig在获奖之后不久,于2014年10月在Science期刊上又发表了一项重量级技术”Lattice light-sheet microscopy”。在接受华盛顿邮报采访时,Betzig教授表示这项技术对于生命科学研究的影响甚至将高于他获诺贝尔奖的超分辨技术(“this development will have more of an impact on biological research than the work that earned him a Nobel Prize”)。我们来了解下什么是Lattice light-sheet microscopy,暂且先翻译为晶格光片显微术。介绍开始之前,同学们需要对light-sheet microscopy(光片显微术)有个简要的认识,与传统荧光显微镜或共聚焦显微镜不同,光片显微镜的照明光轴和检测光轴是垂直的,即下右图橘色light-sheet与物镜光轴是垂直的。而传统荧光显微镜,照明和检测光轴是平行的,成180度,见下图。


为什么我们需要这样特殊的光学架构呢?其一是,这样的光毒性比较小,只有显微成像的样品区域是被激发光照射的,而传统荧光显微镜在焦面以外的样品区域都会被照射激发;其二是,提高Z轴分辨率,因为包括共聚焦技术在内Z轴分辨率一直是受限的。在光片显微镜之前,全内反射荧光显微镜(TIRF)可以将Z轴分辨率提高至100-200nm,但TIRF只能观察靠近容器玻璃片表面的样品。光片显微镜的观察区域比较灵活。比较直观的认识,如果需要进一步提高Z轴分辨率,那么照明光片本身的厚度需要越小越好。这项技术发展的过程中,科学家们在这方面做了很多成功的尝试,从最初用柱面镜(Cylindrical lens)产生光片(SPIM),到后来通过振镜扫描聚焦激光光斑实现更窄的光片(DSLM),以及双光子光斑扫描。前面提到的这几种方法都是用高斯光束(Gaussian beam)来实现的,后来物理学家们发现,用贝塞尔光束(Bessel beam)可以实现更细更窄的光片,来提高Z轴分辨率。上右图,从左至右分别是SPIM, DSLM, 2-photon SPIM, Bessel beam的示意图。

但贝塞尔光束并不是最理想的,尽管它在焦面上确实可以很窄,但是同时会产生非焦平面的激发光。Betzig教授结合了结构光照明(structure illumination microscopy, SIM)的方法,抵消了贝塞尔光束这些负影响,同时也实现了超分辨成像。为了减少成像时间,他们把贝塞尔光束分成七部分,同时扫描,而这样正好形成一个Lattice(晶格)的结构。就这样,Lattice light-sheet把三种领先的技术(即light-sheet, bessel beam, 还有超分辨SIM)结合在一起,实现了活体系统里更高3D光学分辨率、更小光毒性的显微成像技术。



一般来说,Lattice light sheet microscopy系统主要特点如下:
1) 理想样品中,XYZ三维分辨率可实现230 x 230 x 370 nm
2) 照明光片的厚度可以小于600 nm,极大减少光漂白和光毒性
3) 活体样品中可以实现高速成像,300帧/秒
4) 在斑马鱼或果蝇等胚胎中,可实现成像深度50 um


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