Low Power Sub-Wavelength Resolution Fluorescence Imaging

超分辨率是指在低于传统光学成像（例如共聚焦显微镜）所提供的距离尺度上分辨物体和/或观察对比度的能力，其限制在大约照明光波长的一半。在光谱的可见区域，这大约是四分之一到三分之一微米（250- 330nm）。非侵入性亚细胞观察低于这个长度尺度是不可能使用传统的光学显微镜。已经有相当多的学术和商业活动旨在开发揭示100纳米及以下长度尺度结构的技术。这些技术可分为四类：[1]随机重建技术（PALM & STORM）[2]结构照明（SI）[3]受激发射耗尽（STED）技术[4]基态耗尽（GSD）显微镜技术都有严重的缺点。PALM和STORM（分别为光激活定位显微镜和随机光学重建显微镜）需要使用专门的（光激活的）荧光探针，并且通常需要长时间（几个小时）的数据收集。SI使用图案光（复杂光学输入）对样品进行结构化照明，并对所产生的条纹结构进行详细的计算机分析，以提供更高的分辨率，然而，在分辨率增加两倍以上的情况下，需要高输入功率，同时增加了样品损坏的风险。STED通过初始激发光束（PUMP）与耗尽激光（DUMP）（脉冲或连续波）的重叠产生亚微米荧光点，该激光被“塑形”以提供“甜甜圈”强度轮廓。STED的缺点是[a] DUMP波束整形光学器件的费用和复杂性，以及[b]获得高分辨率所需的on - sample DUMP功率。这些对应于光化学损伤和样品加热的开始成为重大风险的强度。GSD显微镜是一种双激光技术，与STED相似，分辨率取决于强度。空间偏移的第二激光器用于（强烈）驱动分子进入长寿命的非荧光三重态，从而减少荧光点。由于淬火（与氧碰撞）导致三重态寿命缩短，GSD分辨率降低，需要开发定制的荧光探针和/或通过专门的安装介质去除氧气。利用中等功率（0.1W）连续波耗尽激光器对脉冲激发后荧光探针发射的时间和空间依赖性进行成像，突破了衍射极限。荧光图像的时间切片可以重新组合，以产生一个图像，显示对比度和结构低于共聚焦显微镜的常规衍射极限。该技术不需要像SI和STED那样复杂的激光束整形。此外，与STED相比，空间分辨率不是由耗竭程度决定的，样品上功率至少比典型的STED甜甜圈低一个数量级。我们将通过在传统的荧光寿命成像显微镜上添加耗尽激光器和开发软件来实现该技术，以分析和重建（修改的）由FLIM系统中常规收集的强度-时空数据提供的信息。该装置将首先用于获得超分辨率的测试结构（20-100nm荧光纳米粒子）和固定细胞中的生物结构。该项目的最后阶段将涉及将该技术应用于活细胞生物过程的研究，涉及与伦敦大学学院细胞与发育生物学小组、伦敦大学学院眼科研究所和伦敦大学学院耳研究所的合作。

Super resolution refers to the ability to resolve objects and/or observe contrast on a distance scale below that afforded by conventional optical imaging (e.g. the confocal microscope) which is restricted to approximately half the wavelength of the illuminating light. In the visible region of the spectrum this is on the order of a quarter to a third of a micron (250- 330nm). Non-invasive sub-cellular observations below this length scale are impossible using conventional optical microscopy . There has been considerable academic and commercial activity aimed at developing techniques that reveal structure on the 100nm length scale and below. These fall into four categories [1] Stochastic Reconstruction Techniques (PALM & STORM) [2] Structured Illumination (SI) [3] Stimulated Emission Depletion (STED) Techniques [4] Ground State Depletion (GSD) Microscopy All have serious drawbacks. PALM and STORM (photo-activated localization microscopy and stochastic optical reconstruction microscopy respectively) require the use of specialised (photo-activatable) fluorescent probes and often long (several hours) data collection. SI uses structured illumination of the sample with patterned light (complex optical input) and detailed computer analysis of the resulting fringe structure to provide increased resolution, yet increases in resolution above a factor of two require high input powers with an increased risk of sample damage. STED creates a sub-micron fluorescent spot by the overlap of the initial exciting beam (PUMP) with a depletion (DUMP) laser (pulsed or continuous wave) which is 'shaped' to provide a 'doughnut' intensity profile. The drawbacks of STED are [a] the expense and complexity of the DUMP beam-shaping optics and [b] the on sample DUMP powers that are required to obtain high resolution. These correspond to intensities where the onset of photochemical damage and sample heating becomes a significant risk. GSD microscopy is a two laser technique and is similar to STED in that resolution is intensity dependent. A spatially offset second laser is used to (strongly) drive molecules into long lived non-fluorescing triplet states resulting in a reduced fluorescent spot. GSD resolution is degraded by triplet lifetime shortening due to quenching (collisions with oxygen) requiring the development of customised fluorescent probes and/or the removal of oxygen by specialised mounting media.Our new technique for super-resolution breaks the diffraction limit through imaging the modifications to the time and spatial dependence of fluorescent probe emission following pulsed excitation using a moderate power (0.1W) continuous wave depletion laser. Time slices of the fluorescent image can be recombined to yield an image which reveals contrast and structure below the conventional diffraction limit of a confocal microscope. The technique does not require sophisticated laser beam shaping as in SI and STED. Also, in contrast to STED spatial resolution is not critically determined by the degree of depletion and on-sample powers will at the very least be an order of magnitude below that of the typical STED doughnut. We will realise the technique by the addition of a depletion laser to a conventional fluorescence lifetime imaging microscope and the development of software to analyse and reconstruct the (modified) information provided by the intensity-space-time data that is routinely collected in FLIM systems. The apparatus will be used firstly to obtain super-resolution in test structures (20-100nm fluorescent nanoparticles) and biological structures in fixed cells. The final phase of the project will involve the application of the technique to the study of biological processes in live cells involving collaborations with UCL groups in Cell & Developmental Biology, The UCL Institute of Opthalmology and the UCL Ear Institute.