Enabling Reliable Testing Of SMLM Datasets

实现 SMLM 数据集的可靠测试

• 批准号: BB/X01858X/1
• 负责人: Susan Cox
• 金额: $ 79.65万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX01858X%2F1
• 关键词: Enabling; Reliable; Testing; SMLM; Datasets

Fluorescence microscopy is a key technology for biomedical research due to its ability to image the distribution of specific proteins in cells. Over the last twenty years the resolution achievable with fluorescence microscopy has been improved from around 200nm down to 20nm. The most popular of these methods, single molecule light microscopy (SMLM), collects a series of images and then processes it to create a reconstructed image of the sample with improved resolution. While this method is experimentally simple and can achieve very good resolution, it has a major drawback: the image processing it relies on can lead to artifacts (false structures) in the reconstructed image that are hard to spot, leading biologists to draw incorrect conclusions about their data. It is extremely difficult to test for these artifacts, and methods to do so have only recently been developed. Here the two leading developers of data assessment for SMLM want to combine forces to create a new tool which enables testing using both of their approaches.We will create an integrated software package that tests for resolution (using a metric improved in accuracy compared to the standard one used) and two types of artifact. The first, artificial sharpening, is common issue encountered in multicolour and live cell SMLM, and any reconstruction method that doesn't find positions of fluorophores, but instead modifies the input images. The second examines how well the SMLM reconstructed image agrees with a widefield image of the sample, and is good at spotting missing structure, variation in the proportion of fluorophores being detected across the image, and issues caused by the background. By testing for all of these different types of artifact, users will be able to have confidence that their data analysis is not introducing errors into their images.Code will be developed on a Github repository and source code will be made available. This is in keeping with the long track record of the applicants in releasing open source software. The software will be released in two different packages: as an ImageJ/Fiji plugin (the most common system used by microscopists and biological/biomedical researchers) and also in napari (a new python-based image processing package, which is increasingly used by methods developers). We will seek to establish our method as a standard test before publication, and encourage the field to develop further extensions as our understanding of SMLM improves, with a process of code review ultimately determining which modifications are accepted.We will also assess a range of publicly available data, and acquire new exemplar datasets to allow us to illustrate to potential users likely artifacts and under what circumstances they might be encountered. While there is an increasing amount of SMLM data available, this usually focuses on high quality raw data which others might want to process. We also want to generate datasets in which raw data likely to produce errors is deliberately generated. The lack of consistent standards and tests contributes to the reproducibility crisis in science: it is impossible to benchmark or validate the data analysis in SMLM studies because there are no agreed standards to which such studies should adhere. Our approach, uniting the best of the current tests available, has the support and confidence of the SMLM developer community and support from facility managers and researchers who rely on SMLM. Setting such a standard for testing will allow journals to finally put in place a consistent set of requirements for SMLM data verification, which is both actually possible for non-specialist researchers to fulfill, and provides rigorous quality checks.

荧光显微镜因其能够对细胞中特定蛋白质的分布进行成像而成为生物医学研究的关键技术。在过去 20 年中，荧光显微镜可实现的分辨率已从 200 nm 左右提高到 20 nm。这些方法中最流行的是单分子光学显微镜 (SMLM)，它收集一系列图像，然后对其进行处理以创建分辨率更高的样品重建图像。虽然这种方法在实验上很简单并且可以实现非常好的分辨率，但它有一个主要缺点：它所依赖的图像处理可能会导致重建图像中难以发现的伪影（错误结构），从而导致生物学家得出错误的结论他们的数据。测试这些伪影极其困难，并且最近才开发出这样做的方法。在这里，SMLM 数据评估的两个领先开发人员希望联合起来创建一个新工具，可以使用他们的两种方法进行测试。我们将创建一个集成软件包来测试分辨率（使用与标准相比精度更高的指标）一种已使用）和两种类型的工件。第一个是人工锐化，是多色和活细胞 SMLM 以及任何无法找到荧光团位置而是修改输入图像的重建方法中遇到的常见问题。第二个检查 SMLM 重建图像与样品的宽场图像的一致性，并且擅长发现缺失的结构、图像中检测到的荧光团比例的变化以及由背景引起的问题。通过测试所有这些不同类型的工件，用户将能够确信他们的数据分析不会在图像中引入错误。代码将在 Github 存储库上开发，并且将提供源代码。这与申请人在发布开源软件方面的长期记录是一致的。该软件将以两个不同的软件包发布：作为 ImageJ/Fiji 插件（显微镜学家和生物/生物医学研究人员最常用的系统）以及 napari（一种新的基于 python 的图像处理软件包，越来越多地被方法使用）开发商）。我们将寻求在发布之前将我们的方法建立为标准测试，并鼓励该领域随着我们对 SMLM 理解的提高而开发进一步的扩展，并通过代码审查过程最终确定接受哪些修改。我们还将公开评估一系列可用数据，并获取新的示例数据集，以便我们向潜在用户说明可能的工件以及在什么情况下可能会遇到它们。虽然可用的 SMLM 数据量不断增加，但这通常侧重于其他人可能想要处理的高质量原始数据。我们还希望生成数据集，其中故意生成可能产生错误的原始数据。缺乏一致的标准和测试导致了科学的可重复性危机：不可能对 SMLM 研究中的数据分析进行基准测试或验证，因为没有此类研究应遵守的商定标准。我们的方法结合了当前可用的最佳测试，得到了 SMLM 开发人员社区的支持和信心，以及依赖 SMLM 的设施经理和研究人员的支持。制定这样的测试标准将使期刊最终能够对 SMLM 数据验证提出一套一致的要求，这对于非专业研究人员来说实际上是可能实现的，并提供严格的质量检查。