Schwenzfeier Jan, Weischer Sarah, Bessler Sebastian, Soltwisch Jens
Institute of Hygiene, University of Münster, 48149 Münster, Germany.
Münster Imaging Network, Cells in Motion Interfaculty Centre, University of Münster, 48148 Münster, Germany.
J Am Soc Mass Spectrom. 2024 Dec 4;35(12):2950-2959. doi: 10.1021/jasms.4c00279. Epub 2024 Oct 9.
We introduce Fluorescence Integrated Single-Cell Analysis Script (FISCAS), which combines fluorescence microscopy with MALDI-MSI to streamline single-cell analysis. FISCAS enables automated selection of tight measurement regions, thereby reducing the acquisition of off-target pixels, and makes use of established algorithms for cell segmentation and coregistration to rapidly compile single-cell spectra. MALDI-compatible staining of membranes, nuclei, and lipid droplets allows the collection of fluorescence data prior to the MALDI-MSI measurement on a timsTOF fleX MALDI-2. Usefulness of the software is demonstrated by the example of THP-1 cells during stimulated differentiation into macrophages at different time points. In this proof-of-principle study, FISCAS was used to automatically generate single-cell mass spectra along with a wide range of morphometric parameters for a total number of roughly 1300 cells collected at 24, 48, and 72 h after the onset of stimulation. Data analysis of the combined morphometric and single-cell mass spectrometry data shows significant molecular heterogeneity within the cell population at each time point, indicating an independent differentiation of each individual cell rather than a synchronized mechanism. Here, the grouping of cells based on their molecular phenotype revealed an overall clearer distinction of the different phases of differentiation into macrophages and delivered an increased number of lipid signals as possible markers compared with traditional bulk analysis. Utilizing the linkage between mass spectrometric data and fluorescence microscopy confirmed the expected positive correlation between lipid droplet staining and the overall signal for triacylglyceride (TG), demonstrating the usefulness of this multimodal approach.
我们引入了荧光集成单细胞分析脚本（FISCAS），它将荧光显微镜与基质辅助激光解吸/电离质谱成像（MALDI-MSI）相结合，以简化单细胞分析。FISCAS能够自动选择紧密的测量区域，从而减少非目标像素的采集，并利用既定的细胞分割和配准算法快速编译单细胞光谱。对细胞膜、细胞核和脂滴进行与MALDI兼容的染色，可以在timsTOF fleX MALDI-2上进行MALDI-MSI测量之前收集荧光数据。通过THP-1细胞在不同时间点刺激分化为巨噬细胞的例子，证明了该软件的实用性。在这项原理验证研究中，FISCAS用于自动生成单细胞质谱以及一系列形态计量参数，这些参数来自于在刺激开始后24、48和72小时收集的总共约1300个细胞。对形态计量学和单细胞质谱数据的综合分析表明，在每个时间点的细胞群体中存在显著的分子异质性，这表明每个细胞是独立分化的，而不是同步机制。在这里，基于分子表型对细胞进行分组，与传统的整体分析相比，更清晰地揭示了分化为巨噬细胞的不同阶段，并提供了更多的脂质信号作为可能的标志物。利用质谱数据与荧光显微镜之间的联系，证实了脂滴染色与甘油三酯（TG）总体信号之间预期的正相关，证明了这种多模态方法的实用性。
