Photochromic rhodamines provide nanoscopy with optical sectioning
Photochromic rhodamines provide nanoscopy with optical sectioning
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DOI：
10.1002/anie.200702167
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Hell, S. W.
中科院分区：
文献类型：
--
作者：
Foelling, J.;Belov, V.;Hell, S. W.
自从1873年阿贝的开创性工作以来，人们普遍认为基于透镜的（远场）光学显微镜的分辨率被限制在所用光波长的一半左右（% λ/2）然而，在20世纪90年代中期，荧光显微镜概念的出现表明，衍射的限制作用可以从根本上克服。这些概念的主要标志是利用荧光标记的状态不仅产生信号，而且打破衍射势垒。事实上，到目前为止，所有成功超越衍射的方法都依赖于选定的分子状态对——具体来说，一个“亮”的分子状态产生信号，一个“暗”的分子状态确保被测量的信号来自一个亚衍射大小的区域。例如，受激发射耗尽显微镜[2]依赖于荧光单线态到（暗）基态的猝灭，通过使用具有零的焦强度分布。因此，除了那些位于零点位置的分子外，所有分子都被“关闭”。这一概念已成功地扩展到荧光蛋白（亚稳态）状态和光致变色有机化合物之间的切换[3,4]。[3,5]在这种情况下，转换发生在（构象）状态之间，其中一种分子能够连续发射荧光光子。这样做的好处是，开关可以在低亮度下进行。利用分子光电开关打破衍射势垒的另一种方法是随机打开，读出荧光，然后关闭孤立的标记分子，这样同时发射（“开”）标记的距离就比显微镜所能分辨的最小距离更远。在这种情况下，荧光的空间限制被定义为单个分子的大小。将来自单个标记的荧光信号成像到相机上，产生一个衍射点，其质心产生发射器的位置，其精度理想情况下仅取决于收集的光子数n和荧光点的全宽半最大值（FWHM）[6]，并近似由FWHM/ ffffffin p给出。分子必须回到暗态，这样才能很容易读出并计算出相邻分子的质心。对大量标记重复此过程，以亚λ/2分辨率重建其分布。与基于零强度的读出模式（RESOLFT）相比，这种单分子读出策略（称为PALM，[7] STORM，[8]和fPALM[9]）的主要优点是标记分子不会被迫经历几个光电转换周期。另一方面，引入了新的需求和限制。荧光“开”状态必须产生足够的光子，以便精确计算质心。同时，单分子方法需要严格控制光激活分子的最大密度，并且还取决于它们在漫射背景下的可靠定位。因此，“开”和“关”状态之间亮度的有限对比以及荧光读出过程中分子的自发激活限制了该方法用于具有低荧光团浓度的薄样品。在此，我们报告了一种新的光致变色罗丹明衍生物，使我们能够克服这些限制。这种易于控制的光开关化合物在单分子条件下具有高荧光量子产率和高光化学稳定性。由此产生的n的急剧增加产生了大约10纳米的平均定位精度。在…
Since the seminal work of Abbe in 1873, it has been commonly assumed that the resolution of a lens-based (farfield) light microscope is limited to about half the wavelength of the light used (% λ/2).[1] However, in the mid-1990s, fluorescence microscopy concepts emerged that demonstrated that the limiting role of diffraction could be fundamentally overcome. The main hallmark of these concepts was to use the states of the fluorescent marker not just for generating the signal, but also for breaking the diffraction barrier. In fact, all the methods that have successfully outperformed diffraction have so far relied on selected pairs of molecular states—specifically, a “bright” one to generate the signal and a “dark” one to ensure that the measured signal stems from a subdiffraction-sized region. For example, stimulated emission depletion microscopy [2] relies on the quenching of the fluorescent singlet state to the (dark) ground state by using a focal intensity distribution featuring a zero. Thus, all molecules are “switched off” except those located at the position of the zero. This concept has been successfully extended to switching between (metastable) states of fluorescent proteins [3, 4] and photochromic organic compounds.[3, 5] In this case, the switching occurs between (conformational) states, in one of which the molecule is able to successively emit fluorescence photons. The benefit is that the switching can be performed at low levels of light. An alternative way of using molecular photoswitching to break the diffraction barrier is to stochastically switch on, read out the fluorescence, and switch off isolated marker molecules such that simultaneously emitting (“on”) markers are further apart than the minimal distance resolved by the microscope. In this case, the spatial confinement of the fluorescence is down to the size of a single molecule by definition. Imaging the fluorescence signal from an individual marker onto a camera produces a diffraction spot whose centroid yields the location of the emitter, with a precision that ideally depends just on the number of collected photons n and on the full-width-half-maximum (FWHM) of the fluorescence spot,[6] and is approximately given by FWHM/ffiffiffin p. After being recorded, the molecules must go back to a dark state so that one is readily able to read out and calculate the centroid of an adjacent one. Repeating this procedure for a multitude of markers reconstructs their distribution with subλ/2 resolution. The main advantage of this single-molecule read-out strategy (known as PALM,[7] STORM,[8] and fPALM [9]) over the zero-intensity-based read-out mode (RESOLFT) is that the marker molecules are not forced to undergo several photoswitching cycles. On the other hand, new requirements and limitations are introduced. The fluorescent “on” state must yield enough photons to allow the precise calculation of the centroid. At the same time, the single-molecule approach requires a strict control over the maximum density of photoactivated molecules, and it also depends on their reliable localization against a diffuse background. Therefore, a finite contrast in brightness between the “on” and “off” states as well as the spontaneous activation of molecules during fluorescence read-out restrict this approach to thin samples with a low fluorophore concentration.Herein, we report a new photochromic rhodamine derivative that has allowed us to overcome these limitations. This readily controllable photoswitchable compound has a high fluorescence quantum yield and high photochemical stability under single-molecule conditions. The resulting dramatic increase in n yields an average localization precision of approximately 10 nm. In …