Introducing FISCAS,\na Tool for the Effective Generation\nof Single Cell MALDI-MSI Data

We introduce Fluorescence Integrated Single-Cell Analysis\nScript\n(FISCAS), which combines fluorescence microscopy with MALDI-MSI to\nstreamline single-cell analysis. FISCAS enables automated selection\nof tight measurement regions, thereby reducing the acquisition of\noff-target pixels, and makes use of established algorithms for cell\nsegmentation and coregistration to rapidly compile single-cell spectra.\nMALDI-compatible staining of membranes, nuclei, and lipid droplets\nallows the collection of fluorescence data prior to the MALDI-MSI\nmeasurement on a timsTOF fleX MALDI-2. Usefulness of the software\nis demonstrated by the example of THP-1 cells during stimulated differentiation\ninto macrophages at different time points. In this proof-of-principle\nstudy, FISCAS was used to automatically generate single-cell mass\nspectra along with a wide range of morphometric parameters for a total\nnumber of roughly 1300 cells collected at 24, 48, and 72 h after the\nonset of stimulation. Data analysis of the combined morphometric and\nsingle-cell mass spectrometry data shows significant molecular heterogeneity\nwithin the cell population at each time point, indicating an independent\ndifferentiation of each individual cell rather than a synchronized\nmechanism. Here, the grouping of cells based on their molecular phenotype\nrevealed an overall clearer distinction of the different phases of\ndifferentiation into macrophages and delivered an increased number\nof lipid signals as possible markers compared with traditional bulk\nanalysis. Utilizing the linkage between mass spectrometric data and\nfluorescence microscopy confirmed the expected positive correlation\nbetween lipid droplet staining and the overall signal for triacylglyceride\n(TG), demonstrating the usefulness of this multimodal approach.