Extending the performance capabilities of isoSTED
Extending the performance capabilities of isoSTED
复制标题
扩展 isoSTED 的性能
DOI：
10.1016/j.bpj.2021.07.005
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发表时间：
2021
影响因子：
3.4
通讯作者：
Galbraith, Catherine G.
中科院分区：
文献类型：
--
作者：
Boehm, Ulrike;Galbraith, Catherine G.
如果你试图用镜头聚焦光线，你很快就会意识到你无法创造一个点。相反，由于光的衍射，你聚焦的光的强度总是更宽更长。所创建的强度模式称为点扩展函数（PSF）。在19世纪，物理学家Ernst Abbe证明了PSF的大小取决于所用光的波长和透镜的集光角度(1)。根据Abbe的说法，当使用单透镜时，PSF的形状将像椭球，其宽度约为焦平面输入光波长的一半，沿光轴的长度为椭球的两到三倍。显微镜的点积角至关重要，因为它的形状最终决定了传统显微镜的分辨率极限。比PSF小的结构看起来与PSF大小相同，并且沿着光轴具有相同的拉长畸变。然而，如果使用两个相反的透镜，PSF的各向异性可以降低。这种排列，被称为4Pi，使光收集角度加倍，并将沿光轴的分辨率提高三到七倍（2-4）。然而，尽管4Pi显微镜可以达到几乎各向同性的分辨率，但它们仍然是衍射有限的。超分辨率显微镜或纳米显微镜也有光的衍射极限。尽管如此，它们在理论上是不受衍射限制的，因为它们利用了分子状态转换，通常在荧光（ON）和非荧光（OFF）状态之间(5)。受激发射耗尽（STED）、可逆荧光饱和光学跃迁（RESOLFT）和光激活定位/随机光学重建显微镜都在时间或空间上以受控或随机的方式使用荧光团的ON和OFF状态来克服衍射极限。STED显微镜的工作原理与共聚焦显微镜相似。然而，在STED中，两个同心和同步的激光束扫描穿过试样。它们控制荧光团在其激发态（ON状态）和基态（OFF状态）之间的过渡，以创建衍射无限的图像。第一束（开开关束）激发荧光团从它们的关到它们的开状态。然后，第二个甜甜圈形状的光束（关闭开关光束）将激发的荧光团推回到它们的关闭状态，然后它们才能发出荧光。只有来自甜甜圈中心的荧光团的信号没有被推回它们的关闭状态才能被检测到。因此，尽管两束光束都有衍射限制，但使用它们来控制荧光团的开和关状态却打破了衍射限制。RESOLFT显微镜的功能类似，但在不同的构象状态之间切换荧光团，这需要较弱的关闭开关光束。然而，获得RESOLFT图像比STED图像需要更长的时间，因为与当前可用的荧光团在构型状态之间切换所需的像素停留时间平均而言，至少比在荧光团的基态和激发态之间切换大一个数量级。尽管超分辨率显微镜带来了进步，但即使这些技术也可以从早期开发的4 π排列中受益，当任务是达到无限衍射的三维（3D）分辨率时。在这里，在4Pi显微镜中创建的令人难以置信的锐利和光效模式/调制，以及双倍光检测已被证明可以显着改善使用STED (6)， RESOLFT(7)和光激活定位/随机光学重建(8,9…
If you try to focus light with a lens, you will soon realize that you cannot create a point. Instead, the intensity of light that you focus will always be wider and longer because of the diffraction of light. The created intensity pattern is called a point spread function (PSF). In the 19th century, the physicist Ernst Abbe was able to show that the size of the PSF depends on the wavelength of the used light and the light collection angle of the lens (1). According to Abbe, when a single lens is used, the PSF will be shaped like an ellipsoid, with a width of approximately half the wavelength of the input light in the focal plane and a length two to threefold that size along the optical axis. The PSF of a microscope is of fundamental importance because its shape ultimately defines the resolution limit of a conventional microscope. Structures smaller than the PSF will appear to be the same size as the PSF and have the same elongated distortion along the optical axis. However, the anisotropy of the PSF can be reduced if two opposing lenses are used. This arrangement, referred to as 4Pi, doubles the light collection angle and increases the resolution along the optical axis by three-to sevenfold (2–4). However, although 4Pi microscopes can reach a nearly isotropic resolution, they remain diffraction-limited. Superresolution microscopes or nanoscopes also experience the diffraction limit of light. Still, they are theoretically diffraction-unlimited because they take advantage of molecular state transitions, typically between a fluorescent (ON) and nonfluorescent (OFF) state (5). Stimulated emission depletion (STED), reversible fluorescent saturable optical transition (RESOLFT), and photoactivation localization/stochastical optical reconstruction microscopes all use the ON and OFF states of fluorophores either in a controlled or random way in time or space to overcome the diffraction limit. A STED microscope operates similarly to a confocal microscope. However, in STED, two concentric and synchronized laser beams are scanned across the specimen. They control the transition of the fluorophores between their excited (ON state) and their ground state (OFF state) to create a diffraction-unlimited image. The first beam (ON switching beam) excites fluorophores from their OFF to their ON state. Then, the second donut-shaped beam (OFF-switching beam) pushes excited fluorophores back to their OFF state before they can emit fluorescence. Only signal from fluorophores in the center of the donut that were not pushed back to their OFF state can be detected. So, although both beams are diffraction-limited, using them to control the ON and OFF state of the fluorophores breaks the diffraction limit. RESOLFT microscopy functions similarly but switches the fluorophores between different conformational states, which requires a less powerful OFF-switching beam. However, it takes longer to acquire an RESOLFT image than an STED image because the pixel dwell times required for switching between conformational states with the currently available fluorophores are, on average, at least one order of magnitude larger than for switching between ground and excited states of fluorophores. Despite the advances provided by superresolution microscopes, even these technologies can benefit from the earlier developed 4Pi arrangement when tasked with reaching diffraction-unlimited threedimensional (3D) resolution. Here, the incredibly sharp and light-efficient patterns/modulations created in 4Pi microscopes, and the doubled light detection have been shown to significantly improve the images collected using STED (6), RESOLFT (7), and photoactivation localization/stochastical optical reconstruction (8, 9 …