Optimized laser foci for imaging MALDI-mass spectrometry

成像MALDI质谱是一种相当新的分析方法，具有巨大的发展潜力。该技术的主要优点是，它允许同时，无标记和横向分辨的组织切片中的许多生物分子的注册。横向分辨率主要由样品上激光光斑的焦点直径定义。目前作为标准实现的20-50 µm之间的分辨率不提供细胞分辨率，因为它在许多生物和医学研究问题中是需要的。一般来说，只有直径小于10 µ m的激光光斑才能超过此边界。然而，真正的质谱分辨率<10 µm的实现受到几个仪器和MALDI工艺相关因素的阻碍。例如，由于迄今尚未很好理解的光斑尺寸效应，聚焦激光光斑的小型化伴随着产生离子所需的单位面积脉冲能量的超比例增加。此外，关于MS性能特性，激光强度分布也起着重要作用，特别是对于小光斑尺寸。使用具有热点的高斯或更强结构化激光束轮廓，如由用于MALDI的Nd：YAG和N2激光器产生的，可以导致强度峰中的分析物碎片增加。只有通过使用均匀平顶激光轮廓，才能在整个照射区域上实现最佳激发条件。由于侧面的陡峭性和由此良好限定的材料喷射，这些轮廓也特别适合于成像MALDI。在激光材料加工和激光医学领域已经成功实施的光束整形技术的基础上，我们提出使用直径在2到10 μ m之间的平顶光束轮廓。在常规MALDI离子源的焦平面中为200 µm。为此，将研究并进一步开发不同的概念，用于成像MALDI-MS，其整体包括常用MALDI激光系统的两种情况，即空间高相干Nd：YAG和空间低相干N2激光。作为质谱仪，将使用Synapt G2-S（沃茨）和类似于QStar（AB Sciex）的仪器。在第二，密切相关的部分的研究项目的基本原理的MALDI过程将调查方面的焦斑直径和强度分布的影响。MALDI离子信号以及激光诱导材料烧蚀将分别使用质谱仪和光声检测装置记录为这些参数和激光注量的函数。从这些研究中可以预期到改善成像MALDI-MS的可能性的重要提示。

Imaging MALDI mass spectrometry is a fairly recent analytical method with enormous development potential. The major advantage of the technique is that it allows for the simultaneous, label-free and laterally-resolved registration of numerous biomolecules in tissue slices. The lateral resolution is largely defined by the focal diameter of the laser spot on the sample. Resolutions between 20-50 µm, currently achieved as standard, do not provide for a cellular resolution as it is required in many biological and medical research issues. Generally, this boundary is only surpassed for laser spots with sub 10 µm-diameters. However, the realization of a true mass spectrometric resolution <10 µm is hampered by several instrumental and MALDI process-related factors. For example, due to a hitherto not well understood spot size effect miniaturization of the focal laser spot is accompanied by an over-proportional increase of the pulse energy per area necessary for the production of ions. Moreover, with respect to the MS performance characteristics also the laser intensity profile plays has a significant role, especially for small spot sizes. The use of Gaussian or stronger structured laser beam profiles with hot spots, like they are produced by Nd:YAG and N2-lasers employed for MALDI, can lead to an increased analyte fragmentation in the intensity peaks. Only by using homogeneous flat top laser profiles can optimal excitation conditions be achieved over the whole irradiated area. Because of the steepness of the flanks and the thereby well-defined material ejection these profiles are also particular well suited for imaging MALDI-MS. This would be even more so if flat top profiles with square dimensions were utilized.Building on beam shaping techniques that are already implemented successfully in the areas of laser material processing and laser medicine we propose to use flat top beam profiles with diameters between 2 and 200 µm in the focal plane of customary MALDI ion sources. For this purpose, different concepts will be investigated and further developed for imaging MALDI-MS that in their entirety encompass the two cases of commonly used MALDI laser systems, namely the spatially high-coherent Nd:YAG and the spatially low-coherent N2-Laser. As mass spectrometers, a Synapt G2-S (Waters) and an instrument similar to the QStar (AB Sciex) will be employed. In a second, strongly linked part of the research project fundamentals of the MALDI process will be investigated with regard to the influence of the focal spot diameter and its intensity profile. MALDI ion signals as well as the laser-induced material ablation will be recorded as function of these parameters and of the laser fluence, using a mass spectrometer and a photoacoustic detection set-up, respectively. Important hints towards possibilities for improving imaging MALDI-MS are expected from these studies.