Developing methods for mass spectral imaging of environmental samples

创造形象的能力是推进科学理解的基础。这在所有尺度上都是正确的--例如，污染地图如何被用来可视化和理解环境威胁是显而易见的。在小范围内，对单个细胞和部分细胞成像的能力也是现代科学的重要组成部分。已经有成熟的技术来对生物样本中的大分子(基因和蛋白质)进行成像，但对于代谢物、金属离子或化学污染物分子等较小的物质来说，这一技术要少得多。一种可以做到这一点的技术是二次离子质谱仪(SIMS)，在这种技术中，一束离子束(带电粒子)横跨样品。这会从它的表面产生二次离子，这些离子被收集并送入质谱仪，质谱仪对它们进行分类和计数。然后，信号可以被解释为不仅给你提供样品的详细图像，而且还提供关于其化学成分的信息。有许多不同类型的SIMS仪器，其中一种是Ion-TOF TOF-SIMS(代表飞行时间SIMS)，它在两种不同的方式下具有非常高的分辨率。首先，探测器(质谱仪)是高分辨率的，因此它可以区分传统仪器会聚集在一起的两种不同的信号。其次，它具有非常高的空间分辨率：它可以轻松地创建单个细胞大小的详细图像，并解析亚细胞结构。这意味着它在研究表面的生物化学方面具有巨大的潜力--例如，穿过细胞或组织的切片。它也可以很好地处理非模式生态相关生物，这对环境研究特别重要。然而，有许多因素可能会影响TOF-SIMS产生的图像的潜在质量。特别是，如何准备样品以进入仪器可能是至关重要的。我们建议通过将TOF-SIMS应用于三个对比鲜明的范例项目来展示TOF-SIMS在环境研究中的多功能性和实用性：1.成像木虱样本中亚细胞分布的金属离子。林虱是常见的陆地无脊椎动物，它们对污染的反应可以告诉我们受污染地点的生态健康状况。两种不同的木虱物种对有毒金属铅和锌的处理方式非常不同，我们将生成详细的地图，准确地显示这些金属在它们的细胞内的位置。我们也可能确定这些金属离子形成什么样的络合物。2.蚯蚓体内纳米粒子的成像。非常小的颗粒-纳米颗粒-的潜在毒性根本不被很好地理解，所以是当前的一个热门研究课题。显然，能够识别它们在生物样本中的最终位置是至关重要的，但这是具有挑战性的。我们将使用TOF-SIMS作为对蚯蚓中纳米颗粒样本的补充分析，这些样本是由一个单独的项目产生的。想象一种独特的蚯蚓代谢物。蚯蚓是一种常见的土壤动物，在维持土壤质量方面起着关键作用。它们会产生一种独特的化学物质，没有人知道到底是为了什么。它存在于所有蚯蚓物种中，因此肯定发挥着重要的生物学作用：我们最好的猜测是，它通过帮助稳定细胞膜，帮助它们在土壤干燥时存活下来。我们将制作一张在蚯蚓细胞中发现这种化学物质的详细地图，这是了解它的作用的关键第一步。因此，我们的项目旨在为在环境研究中使用TOF-SIMS提供原理证明。对于上面的项目1和2，我们将能够直接与钻石同步加速器(一个巨大、复杂的专业设备)获得的成像数据进行比较：我们预计TOF-SIMS将具有许多优势，例如更高分辨率的图像，而成本和复杂性只有它的一小部分。

The ability to create an image is fundamental to advancing scientific understanding. This is true at all scales - for instance, it is immediately obvious how a pollution map could be used to visualize and understand environmental threats. At the small scale as well, the ability to image individual cells and parts of cells is an essential part of modern science. There are well-established techniques to image large molecules (genes and proteins) in a biological sample, but this is much less routine for smaller things such as metabolites, metal ions, or molecules of chemical pollutants. One technique that can do this is secondary ion mass spectrometry (SIMS), in which a beam of ions (charged particles) is played across a sample. This produces secondary ions from its surface, which are collected and fed into a mass spectrometer, which sorts and counts them. The signal can then be interpreted to give you not only a detailed image of the sample, but also information about its chemical composition.There are a number of different kinds of SIMS instrument, and one in particular, the Ion-Tof TOF-SIMS (standing for time-of-flight SIMS) has very high resolution in two different ways. Firstly, the detector (mass spectrometer) is high resolution, so that it can distinguish two different signals that a traditional instrument would lump together. Secondly, it has very high spatial resolution: it can easily create detailed images of something the size of a single cell, and resolve sub-cellular structures. This means that it has immense potential for studying the biochemistry of surfaces - for instance, a slice through a cell or tissue. It can also work just as well with non-model ecologically relevant organisms, particularly important for environmental research. However, there are many factors that could affect the potential quality of images produced by TOF-SIMS. In particular, how you prepare a sample to go into the instrument is likely to be crucial. We propose to demonstrate the versatility and utility of TOF-SIMS for environmental research by applying it to three contrasting exemplar projects:1. Imaging metal ions at a sub-cellular distribution in samples from woodlice. Woodlice are common terrestrial invertebrates, and their responses to pollution can tell us about the ecological health of a contaminated site. Two different woodlouse species handle the toxic metals lead and zinc very differently, and we will generate detailed maps of exactly where these metals are found within their cells. We may also be able to determine what kind of complexes these metal ions form. 2. Imaging nanoparticles in earthworms. The potential toxicity of very small particles - nanoparticles - is not at all well understood, so is a current hot research topic. It's obviously vital to be able to identify where they end up in a biological sample, but this is challenging. We will use TOF-SIMS as a complementary analysis for samples of nanoparticles in earthworms, generated from a separate project.3. Imaging a unique earthworm metabolite. Earthworms are a common soil animal, and play a key role in maintaining soil quality. They produce a unique chemical, and no-one knows exactly what for. It's present in all earthworm species, so must play an important biological role: our best guess is that it helps them to survive desiccation when soil dries out, by helping stabilize membranes. We will produce a detailed map of where this chemical is found in earthworm cells, the first crucial step in understanding just what it does.Thus, our project aims to provide a proof-of-principle for using TOF-SIMS in environmental research. For projects 1 and 2 above, we will be able to make a direct comparison to imaging data acquired (by a collaborating lab) at the Diamond synchrotron (a huge, complex, specialist facility): we expect the TOF-SIMS will have many advantages, such as higher-resolution images, at a fraction of the cost and complexity.