Mass spectrometry imaging to explore molecular heterogeneity in cell culture.

Molecular analysis on the single-cell level represents a rapidly growing field in the life sciences. While bulk analysis from a pool of cells provides a general molecular profile, it is blind to heterogeneities between individual cells. This heterogeneity, however, is an inherent property of every cell population. Its analysis is fundamental to understanding the development, function, and role of specific cells of the same genotype that display different phenotypical properties. Single-cell mass spectrometry (MS) aims to provide broad molecular information for a significantly large number of cells to help decipher cellular heterogeneity using statistical analysis. Here, we present a sensitive approach to single-cell MS based on high-resolution MALDI-2-MS imaging in combination with MALDI-compatible staining and use of optical microscopy. Our approach allowed analyzing large amounts of unperturbed cells directly from the growth chamber. Confident coregistration of both modalities enabled a reliable compilation of single-cell mass spectra and a straightforward inclusion of optical as well as mass spectrometric features in the interpretation of data. The resulting multimodal datasets permit the use of various statistical methods like machine learning-driven classification and multivariate analysis based on molecular profile and establish a direct connection of MS data with microscopy information of individual cells. Displaying data in the form of histograms for individual signal intensities helps to investigate heterogeneous expression of specific lipids within the cell culture and to identify subpopulations intuitively. Ultimately, t-MALDI-2-MSI measurements at 2-µm pixel sizes deliver a glimpse of intracellular lipid distributions and reveal molecular profiles for subcellular domains.

Introducing FISCAS, a Tool for the Effective Generation of Single Cell MALDI-MSI Data.

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.