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首页> 外文会议>Advanced microscopy techniques II >Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences
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Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

机译：基于光片的荧光显微镜（LSFM）减少了光毒作用，并为现代生命科学提供了新的手段

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Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed cell movements during gastrulation, revealed the development during cell migration processes and showed that an LSFM exposes an embryo to 200 times less energy than a conventional and 5,000 times less energy than a confocal fluorescence microscope. Most recently, we implemented incoherent structured illumination in our DSLM. The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate the effect of the specimen on the image formation process at various depths in objects of different age.
机译：大多数光学技术都应用于基本为二维的平面蜂窝系统。然而，生理上有意义的信息取决于细胞环境的形态，机械特性和生物化学。一个单元需要与其他单元的复杂三维关系。然而，观察多细胞生物学标本仍然是一个挑战。标本散射并吸收光，因此探测光的传递和信号光的收集变得效率低下。许多内源性生化化合物也会吸收光并遭受某种降解（光毒性），这会导致标本发生故障。在常规和共聚焦荧光显微镜中，每当一个平面时，整个样本就被照亮。沿光学Z轴记录的图像堆栈因此为每个平面照亮了整个样本一次。因此，细胞被照亮的频率是观察到的10到20倍，钓鱼的次数是100到300倍。通过改变光学装置可以避免这种情况。基本思想是使用光片，这些光片从侧面进入样品并与宽视场荧光显微镜的焦平面重叠。与落射荧光装置相反，这种方位角荧光装置使用两个独立操作的透镜进行照明和检测。光学切片是固有特性，在靠近焦平面的小体积内没有光毒损害或光致漂白。基于光片的荧光显微镜（LSFM）利用了现代相机技术。 LSFM可以使用激光切割机进行操作并用于荧光相关光谱。在最近几年中，LSFM用于记录斑马鱼从32细胞早期到发育后期的亚细胞分辨率和较短的采样周期（60-90秒/堆栈）的发育。记录速度为5个4-Megapixel大帧/秒，动态范围为12-14位。我们跟踪了胃动过程中的细胞运动，揭示了细胞迁移过程中的发育，并表明LSFM使胚胎暴露的能量比常规方法少200倍，比共聚焦荧光显微镜少5,000倍。最近，我们在DSLM中实现了非相干结构照明。强度调制的光片可以动态频率生成，并允许我们估计样品在不同年龄物体的不同深度处对图像形成过程的影响。

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