Your Next Confocal Should Be a Light-Sheet Microscope

Light-Sheet Fluorescence Microscopy

灯场荧光显微镜是长期高间隔(分钟至几天)实时样品成像的首选方法。共聚焦显微镜中使用的大多数模型适用于灯页显微镜。由于其独特的功能,可以在可以用轻型显微镜中获取的样品光谱中包含其他挑战性标本。

Compared to confocal laser scanning and spinning disk confocal microscopy, light-sheet microscopy enables fast, high resolution, true volume, and in-depth imaging with the following major advantages:

低光漂白

光漂白是指由于样品中荧光团分子的光诱导的荧光造成的永久荧光能力丧失。长期暴露于光线,尤其是在延时研究中,会引起光漂白,阻碍检测荧光分子。

References

Harms, G.S., et al. (2001). Autofluorescent proteins in single-molecule research: applications to live-cell imaging microscopy. Biophys. J. 80: 2396-2408.

IM,K.B。等。(2013)。用组合点FRAP和FCS分析的扩散和结合。细胞仪A 89:876-889。

参数

  • InVi SPIM: 62x/1.1NA, 2048 × 2048 pixel, pixel size 100 × 100 nm, light-sheet thickness 2 µm, illumination time per voxel 25 µs
  • Spinning disk confocal: 60x/1.2NA, 2048 × 2048 pixel, pixel size 100 × 100 nm, pinhole diameter 50 µm or 1.5 Airy units, pinhole distance 250 µm, illumination time per voxel 10 µs
  • 点共聚焦:带有10K共振扫描仪的CLSM;60x/1.2NA,200×200像素,像素尺寸250 nm,针孔直径为50 µm或1.5空单元,每个素的照明时间0.5 µs
  • 荧光寿命:2.5 ns
  • Intersystem crossing rate: 2.5·106 s-1
  • Triplet lifetime: 5 µs
  • Bleach rate: 100 s-1 at 1 kW cm-2

比较灯场显微镜,旋转盘和共聚焦显微镜的光漂白率显示,在使用灯页面显微镜时,光漂白的降低。当对单个平面进行成像 @ 100 fps时,效果已经显现,但是当比较40 µm(1 µm步)的堆栈的成像 @ 100 fps时,差异变得特别令人惊讶。

低光毒性

Long-term imaging can have phototoxic effects on the sample, altering the normal behavior of the cells and the whole specimen.

灯表显微镜在有效使用激发光子的情况下突出,从而最大程度地减少了光毒性效应。这有助于防止产生误导性和人为结果。

The study from Jemielita et al. (2013) brings out seemingly imperceptible phototoxic effects induced by long-term exposure to light. The comparison of light-sheet microscopy and spinning disk microscopy images revealed inappropriate bone development in zebrafish due to photo-damage in spinning disk microscopy.

来源文章

Jemielita, M. et al. (2013) Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light-sheet fluorescence microscopy techniques. Biophotonics 6 (11-12): 920-8

High Temporal and Spatial Resolution

灯表显微镜可实现高成像速度,并有可能捕获更高数量的事件。必威官网体育下载这在快速发生的动态过程中特别相关。

A study by Reichmann et al. (2018) carried out at EMBL serves as an example. It shows that during the first cell division in mouse embryos, the maternal and paternal chromosomes remain separated. Only light-sheet microscopy made these findings possible.

来源文章

Reichmann, J. et al. (2018) Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos. Science, published online.

Optical Sectioning

光学切片是指在3D结构内的特定焦平面的清晰图像。良好的Z分辨率可以实现样品的3D重建。

Fluorescence microscopy, e.g. confocal microscopy, spinning disk confocal and light-sheet microscopy, enable optical sectioning. Confocal Microscopy and Spinning Disk Microscopy image a specific focal plane by point scanning the sample and rejecting out of focus fluorescent signal with a pinhole(s). These techniques enable high-resolution image acquisition at the expense of photo-damaging effects and/or high time consumption.

灯页显微镜提供intrinsic optical sectioning由特定的一个特定foc照明al plane. This is achieved by the orthogonal arrangement of the illumination and detection objective lenses as well as the projection of a thin light-sheet on the sample. Intrinsic optical sectioning significantly reduces photo-bleaching and phototoxic effects offers high acquisition speed and the possibility to perform long-term experiments.

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