A Super-resolution multiphoton and dynamic STORM imaging facility

荧光显微镜的最新进展使我们定位和辨别微小物体的能力有了显著的提高。超分辨率是指能够分辨出比之前根据物理定律认为可能的更小或更近的物体的技术。现在可以看到纳米分辨率和精确度的物体，以及活组织中的深度图像，并可以高速检查快速的生物事件。同时实现所有这些改进是光学显微镜的“圣杯”，但事实证明，这实际上很难做到。在莱斯特大学，我们开发了一种名为SuperRAMP的新混合技术，它可以在活组织深处进行高速、多色、多光子、超分辨率成像。超分辨率随机访问多光子显微镜(SuperRAMP)是一种独特的技术，它将图案化照明与数学方法相结合，以非常高的速度以纳米精度精确定位离散对象。使用声波穿过晶体的设备被用来投射红外光的图案，红外光可以比可见光波长更好地穿过样品，并更好地穿透深层组织。这些图案经过数学处理，以产生高分辨率的图像。与标准显微镜相比，SuperRAMP将横向(Xy)和深度(Z)分辨率都提高了2-3倍，绝对横向分辨率为120 nm。它可以以每秒约10帧的速度收集两幅彩色图像，并以每秒数千个样本的速度从超分辨率的兴趣点收集图像。今天没有其他的显微镜可以在活组织中以这样的分辨率、深度和速度工作。SuperRAMP将与名为dSTORM的第二种互补方法相结合，以提供更高的分辨率。DSTORM使用与SuperRAMP显微镜相同的数学技术，但不进行扫描。虽然制作图像需要更长的时间，但它的分辨率更高，达到20纳米。我们将开发一种超分辨率、多用户设备，使莱斯特的研究人员和更广泛的社区能够使用这些令人兴奋的技术。这将帮助我们推动BBSRC资助的现有研究人员在一系列项目中的学术成就，这些项目包括新蛋白质的合成、细胞分裂过程、中枢神经系统中细胞与细胞通讯的机制、发育和行为的神经学基础--包括控制日常节奏的生物钟--在蝗虫、斑马鱼、果蝇、老鼠和小鼠等模型动物系统中。这个设施将支持中部地区(诺丁汉、莱斯特和沃里克)和更远的地方(剑桥)的研究合作，并使我们能够通过开发功能、超分辨率成像的展示案例来增强我们的商业影响力。开发了这项革命性的技术后，我们处于独特的地位，可以建立一个世界领先的功能性、超分辨率成像中心。

Recent advances in fluorescence microscopy have led to significant improvements in our ability to locate and discriminate small objects. Super-resolution is the term used for technologies that can resolve objects that are smaller or closer together than was previously thought possible according to the laws of Physics. It is now possible to see objects with nanometre resolution and precision as well as image at depth in living tissue and examine rapid biological events at high speed. Achieving all of these improvements at the same time is the "holy grail" of optical microscopy but this has proved practically difficult. At the University of Leicester, we have developed a new hybrid technology called SuperRAMP that allows high speed, multicolour, multiphoton, super-resolution imaging deep in living tissues.Super-resolution, Random Access MultiPhoton microscopy (SuperRAMP) is a unique technique that combines patterned illumination with mathematical methods to pinpoint discrete objects with nanometre precision at very high speeds. Devices that use sound waves passing across crystals are used to project patterns of infra-red light, which can pass through specimens better than visible wavelengths of light and produce better penetration into deep tissues. These patterns are processed mathematically to produce high-resolution images. SuperRAMP improves both the lateral (xy) and depth (z) resolution by 2-3-fold compared to standard microscopes to an absolute lateral resolution of 120 nm. It can collect two colour images at speeds of around 10 frames per second and from super-resolved points of interest at thousands of samples per second. No other microscope available today can operate at this resolution, at depth, and at this speed in living tissues. SuperRAMP will be combined with a second, complementary method called dSTORM to provide even higher resolution. dSTORM uses the same mathematical techniques used for SuperRAMP microscopy but without scanning. Although it takes longer to create an image, it has a higher resolution of 20 nm.We will develop a super-resolution, multi-user facility that will make these exciting technologies available to researchers at Leicester and to the wider community. This will help us to drive academic excellence amongst existing BBSRC funded researchers in projects that range from studies of the synthesis of new proteins, the processes of cell division, mechanisms of cell-cell communication in the central nervous system, the neurological bases for development and behaviour - including circadian clocks that control daily rhythms - in model animal systems including locusts, zebra fish, fruit flies, rats and mice.This facility will support research collaborations within the Midlands (Nottingham, Leicester and Warwick) and further afield (Cambridge) and allow us to strengthen our commercial impact by developing a show case facility for functional, super-resolution imaging. Having developed this revolutionary technology, we are uniquely placed to establish a world-leading centre of excellence for functional, super-resolution imaging.