Super-resolution optical microscopy via nonlinear self-focusing

通过非线性自聚焦的超分辨率光学显微镜

• 批准号: EP/I006826/1
• 负责人: Gail McConnell
• 金额: $ 125.95万
• 依托单位: University of Strathclyde
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FI006826%2F1
• 关键词: Super; resolution; optical; microscopy; via

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.

现代生物医学的主要结论是生命活动取决于蛋白质、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，其中一些分子仅存在几个拷贝。不幸的是，标准光学显微镜的分辨率大约低 100 倍，无法看到单个分子。因此，这些仪器迫切需要超分辨率。自 20 世纪 90 年代以来，人们就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，英国超分辨显微镜的数量可能还不到10台，而且影响甚微。原因不仅在于成本高、仪器复杂和商业化迟缓：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) 需要将样本冷冻多次，每个周期包括激活，然后成像直至光蛋白分子子集完全漂白。诸如 STORM 之类的随机方法和结构化照明技术速度缓慢且计算量大，并且在分辨率方面的改进不如以前的方法，至少对于可用的线性光学器件而言是这样。一种简单且廉价的方法来提高非线性光学显微镜的分辨率对于每个生物医学研究人员来说都是一个福音。该提案正是涉及这样一种方法，即使用非线性光学自聚焦。自聚焦是高功率激光源在具有正克尔非线性的介质中传播引起的非线性效应。光源沿传播轴的高强度光功率导致高阶折射率的有效增加。这种修改后的折射率分布就像聚焦透镜一样，最终结果是输入光束在透明材料内自聚焦。自聚焦在光子学中得到了广泛认可，并在克尔透镜锁模中发挥了巨大作用，以开发超短脉冲激光源，例如非线性光学显微镜中使用的激光源。至关重要的是，我们最近证明了这是一种在光学显微镜中产生分辨率更好的图像的方法。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激发光束的自聚焦。该方法的成功实施可以轻松地为现有显微镜仪器的空间分辨率带来革命性的提高。这些显微镜属于多光子激光扫描显微镜，尽管成本高昂，但已经在英国和海外广泛使用。因此，这项研究可能会带来重大的生物医学发现，并大大增加实验室现有设备库存的价值，而不仅仅是在英国。

项目成果

Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells.

• DOI: 10.1038/srep07359
• 发表时间: 2014-12-08
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Amor R;Mahajan S;Amos WB;McConnell G
• 通讯作者: McConnell G

A femtosecond-pulsed tunable optical parametric generator at 1530-1790 nm for label-free third harmonic generation imaging

1530-1790 nm 飞秒脉冲可调谐光参量发生器，用于无标记三次谐波生成成像

• 发表时间: 2015
• 作者: Tragardh

Energy shedding during nonlinear self-focusing of optical beams.

光束非线性自聚焦过程中的能量脱落。

• DOI: 10.1364/oe.21.023459
• 发表时间: 2013
• 期刊: Optics express
• 影响因子: 3.8
• 作者: Travis C

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

• DOI: 10.1371/journal.pone.0147115
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Amor R;McDonald A;Trägårdh J;Robb G;Wilson L;Abdul Rahman NZ;Dempster J;Amos WB;Bushell TJ;McConnell G
• 通讯作者: McConnell G

Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy.

甲醇浸泡可减少多光子激光扫描显微镜中使用的长波长水浸透镜的球差。

• DOI: 10.1364/boe.3.003314
• 发表时间: 2012
• 期刊: Biomedical optics express
• 影响因子: 3.4
• 作者: Norris G