Your Next Confocal Should Be a Light-Sheet Microscope
灯场荧光显微镜
纸张荧光显微镜方法of choice for long-term, high interval (minutes to days) live sample imaging. Most of the models used in confocal microscopy are suitable for light-sheet microscopy. Due to its unique capabilities, additional challenging specimens are included in the spectrum of samples that can be acquired with a light sheet microscope.
与共聚焦激光扫描和旋转磁盘共聚焦显微镜相比，灯页显微镜可以快速，高分辨率，真实体积和深度成像，并具有以下主要优势：
Low Photobleaching
Photobleaching refers to the permanent loss of ability to fluoresce due to light-induced damage of the fluorophore molecules in a sample. Long-term exposure to light, especially in time-lapse studies, induces photobleaching, hindering the detection of the fluorescent molecules.
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., et al. (2013). Diffusion and binding analysed with combined point FRAP and FCS. Cytometry A 89: 876-889.
Parameters
- 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
- Point confocal: CLSM with 10k resonant scanner; 60x/1.2NA, 200 × 200 pixel, pixel size 250 nm, pinhole diameter 50 µm or 1.5 Airy units, illumination time per voxel 0.5 µs
- Fluorescence lifetime: 2.5 ns
- 跨系统越过率：2.5·106 S-1
- Triplet lifetime: 5 µs
- Bleach rate: 100 s-1 at 1 kW cm-2
A comparison of the photobleaching rates of light-sheet microscopy, spinning disk, and confocal microscopy reveals a reduction in photobleaching when working with light-sheet microscopy. The effect is already visible when imaging a single plane @ 100 fps, but the difference becomes particularly astonishing when comparing imaging of a stack of 40 µm (1 µm steps) @ 100 fps.
Low Photo-Toxicity
长期对t成像具有光毒性的影响he sample, altering the normal behavior of the cells and the whole specimen.
Light-sheet microscopy stands out for its effective use of excited photons, which minimizes phototoxic effects. This contributes to prevent the generation of misleading and artificial results.
Jemielita等人的研究。（2013年）提出了长期暴露于光引起的看似不可感知的光毒性作用。灯场显微镜和旋转盘显微镜图像的比较显示，由于旋转盘显微镜的造影损伤，斑马鱼中的骨发育不合适。
文章来源
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
高时间和空间分辨率
Light-sheet microscopy enables high imaging speed and the possibility to capture a higher number of events. This is of particular relevance in fast occurring dynamic processes.
Reichmann等人的研究。（2018年）在EMBL举行。它表明，在小鼠胚胎的第一个细胞分裂期间，母体和父亲染色体保持分离。只有灯页显微镜才能使这些发现成为可能。
文章来源
Reichmann，J。等。（2018）Zygotes中的双主轴形成使父母的基因组在早期的哺乳动物胚胎中与众不同。科学，在线出版。
光学切片
Optical sectioning refers to the generation of clear images of specific focal planes within a 3D structure. Good Z resolution enables the 3D reconstruction of a sample.
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.
Light-Sheet Microscopy offers固有的光学切片通过一个特定焦平面的特定照明。这是通过照明和检测客观镜片的正交布置以及样品上薄灯表的投影来实现的。固有的光学切片显着降低了光泽，光毒性效应可提供高采集速度，并有可能进行长期实验。
