Development of the worlds first Live Cell Nanoscope

自16世纪末发明第一台复合光学显微镜以来，光学显微镜已经取得了几个关键的进步。从那时起，我们已经经历了几次迭代，从增强放大倍数，到提高分辨率(衍射限制)，定向荧光，高信噪比共焦，以及最近的超分辨率成像。就在2014年，诺贝尔化学奖还因“超分辨荧光显微镜的发展”而获奖。2015年，我们推出了一款革命性的超分辨率荧光显微镜，它不仅打破了传统的分辨率限制(阿贝的限制)，还允许非常快速和温和的成像，因此使其成为最先进的超分辨率‘活细胞成像’工具。然而，这台显微镜仍然受限于通常所说的光学显微镜的SIM/ISM分辨率限制，近似为分辨率=(0.61\*？)/(2\*NA)。那么对于排放物来说呢？525 nm，物镜数值孔径(NA)为1.49，最佳分辨率为107 nm。虽然这确实提供了一个窗口来了解在细胞生物学中发生的一些最小的相互作用，但是通过向下分辨纳米尺度(=50 nm)可以获得的信息量是指数级的。目前可用于基于荧光的纳米显微镜的唯一工具(SMLM，STED，CLEM)都需要固定生物标本，因此不允许你看到在关键的生物事件中发生的大量的亚细胞相互作用，例如细胞分裂，蛋白质到蛋白质的相互作用，细胞与病毒和细菌的相互作用，细胞收缩，等……作为这个项目的一部分，我们计划开发的是世界上第一个能够进行活细胞成像的基于荧光的纳米显微镜。还应该注意的是，所开发的仪器不仅适用于科学研究领域；先进的光学显微镜不再局限于科学实验室，正被用于从医学筛选到药物开发的各种临床和工业(药物)应用。这种高速、活的细胞成像纳米显微镜将为医学筛查应用开辟新的可能性，例如诊断血小板颗粒疾病，用于药物开发的高含量筛选(HCS)，例如开发信使核糖核酸疫苗，以及用于高密度原位测序的基因测序，以空间解析RNA/DNA/蛋白质，以开发个性化药物。

Light microscopy has made several key advancements since the invention of the first compound light microscope in the late 16th century. Since then we have moved through several iterations from enhanced magnification, to improved resolution (diffraction limited), targeted fluorescence, high signal to noise confocal and most recently super resolution imaging.As recently as 2014 the Nobel Prize in Chemistry was awarded for 'the development of super-resolved fluorescence microscopy'. In 2015 we launched a revolutionary super resolved fluorescent microscope which not only broke the traditional limits of resolution (Abbe's limit) but also allowed for very fast and gentle imaging, therefore making it the most advanced tool for super resolved 'Live Cell Imaging.'However, this microscope was still limited to what is commonly referred to as the SIM/ISM limit of resolution for a light microscope, which is approximated as Resolution = (0.61\*?)/(2\*NA). So for an emission ? of 525nm and an objective with Numerical Aperture (NA) of 1.49, the best achievable resolution would be 107nm. Whilst this does offer a window into some of the smallest interactions which take place within cell biology, the increase in the amount of information that can be gained from resolving down at the nanoscopy scale (=50nm) is exponential.Currently the only tools available for fluorescence based nanoscopy (SMLM, STED, CLEM) all require the biological specimen to be fixed and therefore do not allow you to see the huge amount of sub cellular interactions which occur during critical biological events such as cell division, protein to protein interaction, cell interactions with viruses and bacteria, cell contraction, etc...What we are proposing to develop as part of this project is the worlds first fluorescent based nanoscope which enables live cell imaging.It should also be noted that the developed instrument would not only be suitable for the scientific research field; advanced light microscopes are no longer confined to scientific laboratories and are being used for a variety of clinical and industrial (pharmaceutical) applications, from medical screening to drug development.Such a high speed, live cell imaging nanoscope would open up new possibilities for screening applications in medical screening such as for the diagnosis of platelet granule disorder, high content screening (HCS) for drug development such as in the development of mRNA vaccines and gene sequencing for high density in-situ sequencing to spatially resolve RNA/DNA/Proteins for development of personalised medicines.