Development of a standing-wave fluorescence microscope with high nodal plane flatness
Development of a standing-wave fluorescence microscope with high nodal plane flatness
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DOI：
10.1046/j.1365-2818.1997.2290789.x
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发表时间：
1997-09-01
期刊：
影响因子：
--
通讯作者：
Horler, H
中科院分区：
文献类型：
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作者：
Freimann, R;Pentz, S;Horler, H
本文报道了一种具有高节面平坦度的驻波荧光显微镜的研制和应用。与传统荧光显微镜中的均匀激发场不同，驻波荧光显微镜使用激光的驻波图案，该图案由交替的平面节点和对准节点组成，通过沿显微镜的轴向移动它可以区分一组不同的荧光结构，它们的轴向间隔可能只是一个波长的一小部分，从而使驻波荧光显微镜能够区分在常规或共焦荧光显微镜中出现的轴向未分辨的结构，当样品的轴向延伸与光波长相当时，驻波荧光显微镜是最强大的，否则多个平面同时照射并且它们的分离是很难实现的，本工作的目的是开发一种新的SWFM仪器，它允许驻波荧光显微镜具有可控的高节面平坦度，早期的SWFM不允许这种受控的平坦度，这阻碍了图像的解释和处理，另一个设计目标是建立一个紧凑、易用的仪器来促进这一新技术的更广泛的应用。所研制的仪器使用一个绿色发射的氦-霓激光作为光源，一个压电可移动的分束器来产生两个相互相干的相对相位可变的激光光束和两个单模光纤来传输这些光束到显微镜，每束光束通过平凸透镜和物镜传递到样品上，其残余曲率会引起波前变形的唯一反射表面是二色性分束器，节面平坦度通过干涉条纹通过类似于光学表面干涉测试的程序来控制，该仪器的性能是使用干燥的和荧光标记的大鼠心肌细胞来测试的，SWFM能够区分轴向分离仅为波长的一小部分的应力纤维层，如此小距离的层将完全位于常规或共焦荧光显微镜的景深内，因此这两种方法无法区分。为了从SWFM图像中获得进一步的信息，将图像用作诸如由Krishnamurthi等人构思的图像处理算法的输入数据将是有利的。SPIE，2655,1996，18-25)。为了最大限度地减少样品引起的节面变形，样品应该埋入折射率相近的介质中。利用本文提出的测量节面平坦度的方法，可以检查折射率的适当匹配。为此，乳胶珠的荧光层将简单地被样品取代，将所开发的SWFM与嵌入在匹配折射率的介质中的样品相结合，并进行进一步的图像处理，将充分发挥驻波荧光显微镜的潜力。
This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness.As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light, This pattern consists of alternating planar nodes and antinodes, By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished, Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope, An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light, Otherwise several planes are illuminated simultaneously and their separation is hardly feasible,The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness, Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing, Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique.The instrument developed uses a green-emitting helium-neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope, Each beam is passed on to the specimen by a planoconvex lens and an objective lens, The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter, Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces,The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats, The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength, Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods,To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al, (Proc. SPIE, 2655, 1996, 18-25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen,A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.