Lipid MS/MS spectra are acquired through tandem mass spectrometry, where lipids are first ionized and then fragmented to produce characteristic ions. Mass-to-charge ratios (m/z) and intensities of these fragment ions are detected and recorded as MS/MS spectra. The spectra are then analyzed using software to match observed fragments with theoretical lipid structures, enabling lipid identification and quantification.

Peptide MS/MS spectra data are mass-to-charge ratio (m/z) values and intensities of fragmented peptides ions. MS/MS spectra are analyzed by matching observed fragment ions to theoretical ions from protein databases, enabling accurate peptide and protein identification. Those data are used to determine the amino acid sequence of peptides, quantify proteins, and characterize post-translational modifications in biological samples.

Image-based approaches, such as Vizgen Merscope, 10xGenomicx Xenium and Nanostring CosmX use microscopy and imaging technologies to visualize and quantify gene expression directly within tissue sections. They rely on fluorescently labeled probes which are designed to target specific mRNA sequences. Those approaches offer a high spatial resolution (subcellular), but for a limited number of targets (several hundreds, up to 5k for Xenium). The segmentation step is crucial to define the cell boundaries and to attribute a particular probe signal to a particular cell and obtain a gene x cell matrix associated to the spatial positions of each cell.

Sequencing-based approaches, such as Visium or Slide-Seq, are based on in situ transcript capture followed by ex-situ sequencing, with the addition of barcodes to link each captured molecule to its original spatial position. Those approaches allow whole-transcriptome gene expression profiling, at the cost of a low resolution dependant of the size of capture spots or barcoded beads.

Whole transcriptome single-cell gene expression. This assay can be obtained through several technologies and protocols, depending on the experimental design, goals to achieve, the required readout and sensitivity.

Whole transcriptome gene expression, obtained by the aggregation of RNA from many cells to provide an average expression profile for an entire sample. Classically, this assay is obtained using short-read Illumina sequencing platforms