In MSI, spectra are acquired point by point, and the ionization efficiency of each pixel is affected by local matrix, surface topography and salt content, making it difficult to perform absolute calibration with a consistent solution environment as in LC-MS; thus the intensity of the same m/z across different pixels reflects relative abundance rather than absolute concentration.
In other words, MSI excels at relative conclusions such as 'region A has a higher signal than region B,' whereas a statement like 'region A has a concentration of exactly 12.3 ng/mg' requires additional calibration. This is the essential difference between 'seeing the distribution' and 'measuring the concentration.'
Absolute quantitation requires introducing internal standards (e.g., isotope-labeled analyte) and matching tissue matrix effects to build pixel-level calibration curves; common strategies include adding a uniform internal-standard layer, using adjacent homogenate LC-MS as an anchor, or embedding quantitative standards into the section.
Even so, tissue heterogeneity, ionization suppression and scan drift all introduce error, so absolute-quantitation MSI is mostly used in methodologically mature internal-standard scenarios (such as pharmacokinetics) rather than applied casually.
In spatial biology, the vast majority of scientific questions (target-site enrichment, tumor vs. adjacent tissue, metabolic heterogeneity) do not require absolute concentration but rather trustworthy relative distribution and inter-group differences—precisely MSI's strength.
In new-drug development, MSI often complements homogenate LC-MS quantitation: LC-MS gives overall exposure (absolute quantitation) while MSI gives spatial localization (relative distribution); only by combining the two do you know both 'how much' and 'where'.
If you only need distribution and differences, semi-quantitation suffices, with the focus on proper normalization and registration; if absolute quantitation is required, design internal-standard and calibration experiments, perform method validation, and anchor with LC-MS wherever possible.
For any kind of quantitation, the molecular images must be registered with H&E / immunohistochemistry so that 'signal intensity' can be translated into 'histological meaning.' Neo-Source LDPI/DPI's matrix-free ambient route reduces sample-preparation variables and helps improve the repeatability of quantitation.
To obtain detailed specifications, compatible models, or a quotation for the MSI LDPI / DPI full series imaging ion sources, visit the Neo-Source official website, or contact the official team for compatibility advice tailored to your mass spectrometer (Agilent / SCIEX / Thermo and other mainstream MS).