Optical Superresolution Microscopy (Nanoscopy)

光谱学在“衍射极限”以下的延伸（约三分之一） 光的波长;例如，230 nm）近年来已通过两种不同的 显微镜类别：“PALM/STORM”和“RESOLFT/STED”。 前者再造了一个生物 场景在一个'pontillist'的方式;中心的个别荧光'油漆'点位于 在现场以20纳米的精度，一次几个，直到完整的画面出现。 是 精确但极其缓慢。 第二种方法，STED（模拟排放损耗） 用另一个漫射“圆环”光束叠加照亮场景的正常光斑， 工作是消除边缘的荧光。 这在中心留下了一个较小的点 扫描整个图像，以50 nm的细节显示它。 两种类型的 显微镜是可商购的。 PALM版本价格便宜，但速度慢，最适合 获取静止图像。 STED版本（超过110万美元）具有视频纳米显微镜的潜力 但是施加大的激光功率（在“擦除”环形光束中），这会损害活细胞。 我们的大部分纳米研究工作都致力于STED和类似STED的方法。 我们已经构建 我们自己的STED显微镜围绕现有的汽车激光器和FCS检测电子设备（来自其他 前项目）。 我们设计了（并暂时获得专利）一个'azicon'（方位角 以使环形光束的中心光斑非常暗（保持中心光斑 图像中的亮度，允许更强的擦除光束，因此允许更精细的分辨率）。 为了广泛使用STED，我们为STED染料开发了一种通用的校准方案，使纳米学家能够补偿（在数据处理过程中）他们各自的光学器件或激光器中的怪癖。“饱和度强度”校准手稿先前已发表。 近年来，我们设计了一种新型的 提供两个关键特征的荧光染料：1.擦除束的功率要求较低。 这使得在活细胞中进行更精细的分辨率和更长时间的观察成为可能， 更实际。2.同步擦除光束。 STED以前仅限于两个 颜色，但我们的染料固有的机制扩大了可用的调色板。 这是 重要的是为将要绘制的大分子图像提供生物背景。 多色微管蛋白原纤维和珠已经在同一个单帧图像中成像。 我们还开始利用我们的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CW和ns脉冲，并且我们致力于将外部“TAQ”天线添加到流行的荧光蛋白“油漆”分子的GFP家族中。后者很受欢迎，因为它们可以与细胞中感兴趣的结构遗传相关。 最后，结合STAQ或STED与脉冲调制的甜甜圈时间分布已在理论上进行了研究，建议甚至更低的功率编码，我们已经设计了一个“STEN”（“时空编码纳米显微镜”）显微镜计划的新STED显微镜。实现这一点的软件处于早期开发阶段。 基于与其他人的“PIM”方法类似的多光子光漂白/搁置的进一步发展正在进行中，需要我们建立一个多光束定时照明仪器，该仪器由于现场存在有限而被延迟。 我们还开始建立一个锁定放大器baser SE（受激发射）显微镜，其光谱应该帮助我们完善我们的STAQ方法和探针。 .

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 in early development stages. 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. .