Low Power Sub-Wavelength Resolution Fluorescence Imaging

超分辨率是指在距离尺度上分辨物体和/或观察对比度的能力，所述距离尺度低于常规光学成像（例如共焦显微镜）所提供的距离尺度，所述常规光学成像被限制为照明光的波长的大约一半。在可见光谱区，这是在四分之一到三分之一微米（250- 330 nm）的数量级。使用传统的光学显微镜不可能对低于该长度尺度的亚细胞进行非侵入性观察。有相当多的学术和商业活动旨在开发揭示100 nm及以下长度尺度结构的技术。这些分为四类[1]随机重建技术（PALM和STORM）[2]结构照明（SI）[3]受激发射耗尽（STED）技术[4]基态耗尽（GSD）显微镜所有这些都有严重的缺点。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（可光激活的）荧光探针，并且通常需要长时间（数小时）的数据收集。SI使用具有图案化光（复杂的光学输入）的样品的结构化照明和所得条纹结构的详细计算机分析来提供增加的分辨率，然而分辨率增加到两倍以上需要高输入功率，具有增加的样品损坏风险。STED通过初始激发光束（E10）与耗尽（DUMP）激光（脉冲或连续波）的重叠产生亚微米荧光点，其“成形”以提供“甜甜圈”强度分布。STED的缺点是[a] DUMP光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样品DUMP功率。这些强度对应于光化学损伤和样品加热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相似之处在于分辨率取决于强度。空间偏移的第二激光器用于（强烈地）驱动分子进入长寿命的非荧光三重态，导致减少的荧光点。GSD分辨率因猝灭导致的三重态寿命缩短而降低我们的超分辨率新技术突破了衍射极限，通过使用中等功率的脉冲激发对荧光探针发射的时间和空间依赖性进行成像，（0.1W）连续波耗尽型激光器。荧光图像的时间片可以被重新组合以产生图像，该图像揭示了低于共聚焦显微镜的常规衍射极限的对比度和结构。该技术不需要像SI和STED中那样复杂的激光束成形。此外，与STED相反，空间分辨率不是由耗尽程度决定的，并且采样功率将至少比典型STED甜甜圈低一个数量级。我们将实现的技术，通过添加一个耗尽激光到传统的荧光寿命成像显微镜和软件的开发，以分析和重建（修改）的信息所提供的强度-空间-时间的数据，定期收集在FLIM系统。该装置将首先用于在测试结构（20- 100 nm荧光纳米颗粒）和固定细胞中的生物结构中获得超分辨率。该项目的最后阶段将涉及将该技术应用于活细胞中生物过程的研究，涉及与UCL细胞与发育生物学小组，UCL眼科研究所和UCL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.