Super-resolution optical microscopy via nonlinear self-focusing

现代生物医学的主要结论是，生命活动依赖于蛋白质、碳水化合物和脂质分子之间的特定相互作用。在单个细胞中，有数千种不同类型的分子，有些分子仅以几个拷贝的形式存在。不幸的是，标准光学显微镜的分辨能力大约差100倍，无法看到单个分子。因此，在这些仪器中迫切需要超分辨率。自20世纪90年代以来，已经提出了实现超分辨率的方法，并带来了希望。然而，到目前为止，英国的超分辨率显微镜的数量可能不到10台，而且它们几乎没有影响。原因不仅是高成本，仪器复杂和商业化缓慢：每种方法都有严重的实际缺点。例如，受激发射损耗（STED）显微镜需要使用特殊的荧光团和复杂的多波长激光源。光激活显微镜（PALM）需要通过许多循环冷冻标本，每个循环包括激活，然后成像到光蛋白分子子集的完全漂白。诸如STORM和结构化照明技术的随机方法是缓慢的并且计算密集的，并且至少在可用的线性光学器件的情况下不提供与先前方法一样大的分辨率改进。一种简单而廉价的方法来提高非线性光学显微镜的分辨率对每个生物医学研究人员来说都是一个布恩。自聚焦是高功率激光源在具有正克尔非线性的介质中传输时产生的一种非线性效应。光源沿着传播轴的高量级光功率导致高阶折射率的有效增加。然后，该修改的折射率分布像聚焦透镜一样起作用，并且最终结果是输入光束在透明材料内的自聚焦。自聚焦在光子学中得到了很好的认可，并且在克尔透镜锁模中发挥了很大的作用，以开发超短脉冲激光源，例如用于非线性光学显微镜的那些激光源。至关重要的是，我们最近已经证明这是一种在光学显微镜中产生更好分辨率图像的方法。常见的浸没介质（空气、水、油）具有非常低的正克尔非线性，并且考虑到非线性光学显微镜中通常采用的激光参数，不满足自聚焦阈值条件。然而，计算和初步的实验研究表明，某些有机水溶性化合物可能具有足够高的克尔非线性，以支持自聚焦的激发光束。这种方法的成功实施可以很容易地带来革命性的改善空间分辨率的现有显微镜仪器。这些是被称为多光子激光扫描显微镜的类型，并且已经在英国和海外广泛使用，尽管它们的成本很高。因此，这项研究可能会导致重大的生物医学发现，并大大提高实验室现有设备的价值，不仅在英国。

The main conclusion of modern biomedical science is that the activities of life depend on specific interactions between protein, carbohydrate and lipid molecules. In a single cell there are thousands of different types of molecules, some presented as only a few copies. Unfortunately, the resolving power of a standard optical microscope is approximately 100 times too poor to see individual molecules. Super-resolution is therefore desperately needed in these instruments.Methods for achieving super-resolution have been proposed since the 1990's and have raised hopes. However, the number of super-resolving microscopes in the UK is, so far, probably less than 10, and they have had little impact. The reasons are not only high cost, instrumental complexity and tardy commercialisation: each method has serious practical disadvantages. For example, stimulated emission depletion (STED) microscopy requires the use of special fluorophores and sophisticated multi-wavelength laser sources. Photo-activation microscopy (PALM) needs the specimen to be frozen through many cycles, each cycle consisting of activation and then imaging to the full bleaching of a subset of photo-protein molecules. Stochastic methods such as STORM and structured illumination techniques are slow and computationally intensive and do not provide as large an improvement in resolution as the previous methods, at least with the available linear optics. A simple and inexpensive method to increase the resolution in nonlinear optical microscopy would be a boon to every biomedical researcher.This proposal concerns just such an approach, using nonlinear optical self-focusing. Self-focusing is a nonlinear effect caused by the propagation of a high-power laser source in a medium with a positive Kerr nonlinearity. The high-magnitude optical power of the light source along the propagation axis causes an effective increase in the higher order refractive index. This modified refractive index distribution then acts like a focusing lens and the net result is self-focusing of the input beam within the transparent material. Self-focusing is well recognised in photonics and is employed to great effect in Kerr lens mode-locking to develop ultra-short pulsed laser sources such as those used in nonlinear optical microscopy. Crucially, we have recently demonstrated this as a means for producing better-resolved images in an optical microscope. Common immersion media (air, water, oil) have very low positive Kerr nonlinearity and considering the laser parameters typically employed in nonlinear optical microscopy, the self-focusing threshold condition is not met. However, calculations and preliminary experimental studies show that certain organic water-soluble compounds may have sufficiently high Kerr nonlinearity to support self-focusing of the excitation beam. The successful implementation of this method could easily bring about a revolutionary improvement in spatial resolution of pre-existing microscope instrumentation. These are of the type known as multi-photon laser scanning microscopes, and are already widespread in the UK and overseas, in spite of their high cost. Consequently, this research could lead to major biomedical discoveries and add vastly enhanced value to the existing equipment stock of laboratories, not only in the UK.