Constructing a cost-efficient, high-throughput and high-quality single-molecule localization microscope for super-resolution imaging
Constructing a cost-efficient, high-throughput and high-quality single-molecule localization microscope for super-resolution imaging
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DOI：
10.1038/s41596-022-00730-6
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发表时间：
2022-08-24
期刊：
影响因子：
14.8
通讯作者：
Klenerman, David
中科院分区：
文献类型：
--
作者：
Danial, John S. H.;Lam, Jeff Y. L.;Klenerman, David
该协议提供了一个循序渐进的指导，以建立一个负担得起的单分子定位显微镜设置的高通量超分辨率成像，通过使用现成的和机械加工的零件。单分子定位显微镜（SMLM）利用现代光学的力量，释放超精密的结构纳米复杂的生物机器在其原生环境，以及超灵敏和高分辨率的成像。具有单分子灵敏度的高通量医学诊断。为了实现这种非凡的速度和分辨率，SMLM装置要么由具有强大光学工程专业知识的研究实验室建造，要么以高昂的价格出售。由于技术或财政原因，生命科学家无法获得SMLM，这不利于依赖于超分辨率成像的生物和生物医学发现的进展。在这项工作中，我们提出了NanoPro，一种经济，高通量，高质量和易于组装的SMLM，用于超分辨率成像。我们表明，我们的仪器的性能类似于最昂贵的，一流的商业显微镜和竞争对手现有的开源显微镜以较低的价格和结构复杂性。为了促进其广泛采用，我们编写了一个循序渐进的协议，并附有大量的插图，以帮助没有经验的研究人员构建NanoPro，并通过对小至20 nm的地面真实样本进行成像来评估其性能。详细的视觉说明使在显微镜工程方面缺乏专业知识的学生能够构建，验证和使用NanoPro。
This protocol provides a step-by-step guide to building an affordable single-molecule localization microscopy setup for high-throughput super-resolution imaging by using off-the-shelf and machined parts.Single-molecule localization microscopy (SMLM) leverages the power of modern optics to unleash ultra-precise structural nanoscopy of complex biological machines in their native environments as well as ultra-sensitive and high-throughput medical diagnostics with the sensitivity of a single molecule. To achieve this remarkable speed and resolution, SMLM setups are either built by research laboratories with strong expertise in optical engineering or commercially sold at a hefty price tag. The inaccessibility of SMLM to life scientists for technical or financial reasons is detrimental to the progress of biological and biomedical discoveries reliant on super-resolution imaging. In this work, we present the NanoPro, an economic, high-throughput, high-quality and easy-to-assemble SMLM for super-resolution imaging. We show that our instrument performs similarly to the most expensive, best-in-class commercial microscopes and rivals existing open-source microscopes at a lower price and construction complexity. To facilitate its wide adoption, we compiled a step-by-step protocol, accompanied by extensive illustrations, to aid inexperienced researchers in constructing the NanoPro as well as assessing its performance by imaging ground-truth samples as small as 20 nm. The detailed visual instructions make it possible for students with little expertise in microscopy engineering to construct, validate and use the NanoPro in