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.

Visualization of Differential Cardiolipin Profiles in Murine Retinal Cell Layers by High-Resolution MALDI Mass Spectrometry Imaging.

The precise fatty acyl chain configuration of cardiolipin (CL), a tetrameric mitochondrial-specific membrane lipid, exhibits dependence on cell and tissue types. A powerful method to map CL profiles in tissue sections in a spatially resolved manner is matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). To build on and explore this potential, we employed a quadrupole time-of-flight mass spectrometer along with optimized sample preparation protocols. We imaged the CL profiles of individual murine retinal cell layers at a pixel size of 10 µm. In combination with tandem MS, we obtained detailed insights into the CL composition of individual retinal cell layers. In particular, we found differential expression of the polyunsaturated fatty acids (PUFA) linoleic, arachidonic, and docosahexaenoic acids. PUFAs are prone to peroxidation and hence regarded as critical factors in development and progression of retinal pathologies, such as age-related macular degeneration (AMD). The ability of MALDI-MSI to provide cues on the CL composition in neuronal tissue with close to single-cell resolution can provide important insights into retinal physiology in health and disease.

Transmission-Mode MALDI Mass Spectrometry Imaging of Single Cells: Optimizing Sample Preparation Protocols.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) makes it possible to simultaneously visualize the spatial distribution of dozens to hundreds of different biomolecules (e.g., phospho- and glycolipids) in tissue sections and in cell cultures. The implementation of novel desorption and (post-)ionization techniques has recently pushed the pixel size of this imaging technique to the low micrometer scale and below and thus to a cellular and potentially sub-cellular level. However, to fully exploit this potential for cell biology and biomedicine, sample preparation becomes highly demanding. Here, we investigated the effect of several key parameters on the quality of the sample preparation and achievable spatial resolution, that include the washing, drying, chemical fixation, and matrix coating steps. The incubation of cells with formalin for about 5 min in combination with isotonic washing and mild drying produced a robust protocol that largely preserved not only cell morphologies, but also the molecular integrities of amine group-containing cell membrane phospholipids (phosphatidylethanolamines and -serines). A disadvantage of the chemical fixation is an increased permeabilization of cell membranes, resulting in leakage of cytosolic compounds. We demonstrate the pros and cons of the protocols with four model cell lines, cultured directly on indium tin oxide (ITO)-coated glass slides. Transmission (t-)mode MALDI-2-MSI enabled on a Q Exactive plus Orbitrap mass spectrometer was used to analyze the cultures at a pixel size of 2 µm. Phase contrast light microscopy and scanning electron microscopy were used as complementary methods. The protocols described could prove to be an important contribution to the advancement of single-cell MALDI imaging, especially for the characterization of cell-to-cell heterogeneities at a molecular level.