Optical Superresolution Microscopy (Nanoscopy)

光学光谱学在“衍射极限”(约三分之一)以下的延伸 光的波长；例如230 nm)在最近几年通过两个不同的 显微镜类别：“Palm/STORM”和“RESOLFT/STED”。前者重现了一种生物 场景以一种‘庞蒂里斯特’的方式；单个荧光‘油漆’点的中心被定位 以20纳米的精度显示在场景中，一次几个，直到全景显现。它是 很精确，但非常慢。 第二种方法，STED(受刺激排放耗竭) 将照亮场景的法线光点与另一个漫反射“甜甜圈”光束叠加，其 工作就是擦除边缘周围的荧光。这就在中心留下了一个较小的点 扫描图像，以50纳米的细节显示甜甜圈。两种类型的 显微镜可以在商业上买到。Palm版价格便宜，但速度慢，最适合 获取静止图像。STED版本(超过110万美元)具有视频纳米技术的潜力 但是使用强大的激光能量(在“擦除”甜甜圈光束中)，破坏活细胞。 我们的大部分纳米研究工作都致力于STED和类似STED的方法。我们已经建造了 我们自己的STED显微镜围绕着现有的汽车激光和FCS检测电子设备(来自其他 以前的项目)。我们已经设计(并暂时获得专利)一种‘azcon’(方位角 偏振器轴标)以使甜甜圈光束的中心光点非常暗(保留中心 图像的亮度，允许更强的擦除光束，因此分辨率更高)。 为了广泛使用STED，我们开发了一种STED染料的通用校准方案，使纳米显微镜能够补偿(在数据处理期间)他们各自光学或激光中的奇特现象。《饱和强度》校准稿此前已发表。 近年来，我们已经设计、申请了专利，并开始测试一类新的 荧光染料提供了两个主要特点：1.对擦除光束的较低功率要求。 这允许在活细胞中进行更精细的分辨率和更长时间的观察，从而实现视频纳米技术 更切合实际。2.多色同时擦除光束。STED之前被限制为两个 颜色，但我们的染料固有的机制扩大了可用的调色板。这是 在为将要绘制的大分子的图像提供生物学背景方面很重要。 多色的微管蛋白纤维和珠子已经在同一张单帧图像中成像。我们还开始利用我们染料的纳秒性质来设计一种显微镜，使用廉价的二极管激光器来实现我们的STAQ纳米显微镜，包括连续脉冲和ns脉冲，我们还致力于在广受欢迎的GFP荧光蛋白质“油漆”分子家族中添加外部“Taq”天线。后者之所以受欢迎，是因为它们可以从基因上与细胞中感兴趣的结构联系在一起。 最后，将STAQ或STED与脉冲调制的甜甜圈时间分布相结合进行了理论研究，提出了更低的编码功率，并为新的STED显微镜设计了一种“STEN”(“时空编码纳米”)显微镜方案。实现这一目标的软件仍处于早期开发阶段。我们已经恢复了一些遗留的全局代码来适应这项任务。 基于多光子的光漂白/搁置的进一步开发工作正在进行中，这种方法类似于其他“PIM”方法，需要我们建造一台多光束定时照明仪器，但由于现场存在有限，这一仪器已被推迟。 我们还开始建造锁定放大器基SE(受激发射)显微镜，它的光谱应该有助于我们改进STAQ方法和探测器，通过检测Taq天线的暗状态。 。

The extension of optical spectroscopy below the "diffraction limit" (about a third of the wavelength of light; e.g.,230nm) has been realized in recent years by two different classes of microscope: "PALM/STORM" and "RESOLFT/STED". The former recreates a biological scene in a 'pontillist' manner; centers of individual fluorescent 'paint' dots are located with 20nm precision on the scene, a few at a time, until the full picture emerges. It is precise but painstakingly slow. The second method, STED (STimulated Emission Depletion) superposes the normal spot illuminating the scene with another diffuse "donut" beam whose job is to erase fluorescence around the edges. This leaves a smaller spot at the center of the donut to sweep across the image, revealing it in 50nm detail. Both sorts of microscope are commercially available. The PALM version is inexpensive but slow, best for acquiring still images. The STED version (over $1.1M) has the potential for video nanoscopy but applies large laser powers (in the "erase" donut beam) that damages living cells. Most of our nanoscopy effort is devoted to STED and STED-like methods. We have constructed our own STED microscope around existing CARS lasers and FCS detection electronics (from other prior projects). We have designed (and provisionally patented) an 'azicon' (azimuthal polarizer axicon) to make the central spot of the donut beam very dark (preserving central brightness in the image, allowing for stronger erase beams and hence finer resolution). For widespread STED use, we developed a general calibration scheme for STED dyes that enables nanoscopists to compensate (during data processing) for the quirks in their individual optics or lasers. The "Saturation Intensity" calibration manuscript was published previously. In recent years we have designed, patented, and begun testing a new class of fluorescent dyes that provide two key features: 1. lower power requirements for erase beam. This allows finer resolution and longer observations in living cells, making video nanoscopy more practical. 2. Simultaneous multicolor erase beam. STED had previously been limited to two colors, but the mechanism inherent in our dyes expands the available palette. This is important in providing biological context to the image of macromolecules one will paint. Multicolor tubulin fibrils and beads have been imaged in the same, single-frame image. We also began exploiting the nanosecond nature of our dyes to design a microscope using inexpensive diode lasers to achieve our STAQ nanoscopy, both CW and ns-pulsed, and we worked toward adding external "TAQ" antennae to the popular GFP family of fluorescent protein "paint" molecules. The latter are popular because they can be genetically connected to the structure of interest in cells. Finally, combining STAQ or STED with pulse-modulated donut time profiles has been theoretically examined to suggest even lower powers for encoding, and we have designed a "STEN" ("SpatioTemporal Encoding Nanoscopy") microscopy scheme for the new STED microscopes. The software to accomplish this is still in early development stages. We have recovered some legacy global code to adapt to this task. A further development based on photobleaching/shelving with multiple photons similar to the "PIM" methods of others is in process, requiring our building a multibeam timed illumination instrument, which has been delayed by limited on-site presence. We have also begun building a lock-in amplifier baser SE (Stimulated Emission) microscope whose spectra should help us refine our STAQ method and probes, by sensing dark states of TAQ antennae. .