Confocal microscopy on the beamline: novel three-dimensional imaging and sample positioning.
Confocal microscopy on the beamline: novel three-dimensional imaging and sample positioning.
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DOI：
10.1107/s002188981203470x
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发表时间：
2012-10
影响因子：
6.1
通讯作者：
I. Khan;R. Gillilan;I. Kriksunov;R. Williams;W. Zipfel;U. Englich
中科院分区：
文献类型：
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作者：
I. Khan;R. Gillilan;I. Kriksunov;R. Williams;W. Zipfel;U. Englich
共聚焦显微镜，一种已广泛应用于细胞生物学研究的技术，也可应用于蛋白质晶体的可视化和三维成像在高分辨率的同步加速器光束线。使用适于低温使用的市售共聚焦显微镜检查蛋白质晶体样品。一个初步的测试使用自定义共焦设计适用于光束线使用。共焦光学系统的配置是兼容的非线性成像技术，如双光子激发荧光成像和二次谐波产生。这种方法的可能性，探讨使用两种模式：荧光和反射共焦。在荧光模式下，通过浸泡或生长条件将少量染料引入晶体中。在这样的条件下，蛋白质晶体很容易从盐和无定形沉淀物中分离出来，而盐和无定形沉淀物通常不吸收染料。反射模式，这不需要染料，仍然表现出更大的分辨率和灵敏度比传统的宽视场显微镜作为共焦光学配置的结果，表面的细节。该方法固有的三维性质意味着轴上样品视图（沿着X射线束的方向）可以从离轴配置重建，简化了束线设置并提供了低温冷却晶体的独特详细视图。
Confocal microscopy, a technique that has been extensively applied in cellular biological studies, may also be applied to the visualization and three-dimensional imaging of protein crystals at high resolution on synchrotron beamlines. Protein crystal samples are examined using a commercially available confocal microscope adapted for cryogenic use. A preliminary test using a custom confocal design adapted for beamline use is also presented. The confocal optics configuration is compatible with nonlinear imaging techniques such as two-photon excited fluorescence imaging and second harmonic generation. The possibilities of this method are explored using two modes: fluorescence and reflection confocal. In fluorescence mode, small amounts of dye are introduced into the crystal through soaking or growth conditions. Under such conditions, protein crystals are easily resolved from salts and amorphous precipitates, which do not generally take up dye. Reflection mode, which does not require dye, still exhibits greater resolution and sensitivity to surface detail than conventional wide-field microscopy as a result of the confocal optics configuration. The inherent three-dimensional nature of the method means that on-axis sample views (along the direction of the X-ray beam) can be reconstructed from an off-axis configuration, simplifying the beamline setup and providing uniquely detailed views of cryogenically cooled crystals.