Comprehensive Label-Free Expansion Mass Spectrometry Imaging Platform for Subcellular Molecular Visualization at Optical-Level Resolution

Achieving nanometer-scale resolution in label-free molecular imaging while
maintaining molecular integrity is a critical challenge in cellular biology and
neuroscience. Existing imaging methods face notable limitations: optical microscopy
lacks specificity for lipids, Label-free microscopy provides only general class-level
chemical information without identifying individual molecules, and electron microscopy,
though it offers ultra-high spatial resolution, cannot provide molecular information.
Hard ionization techniques like SIMS and LDI achieve fine spatial detail but cause
extensive molecular fragmentation, which limits the detection of endogenous molecules.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI)
offers a promising solution by using a soft ionization process that preserves molecular
structures and provides broad molecular coverage. However, traditional MALDI MSI is
typically limited to spatial resolutions of 5 to 20 micrometers, which is insufficient for
capturing subcellular details.
This proposal seeks to create a comprehensive, label-free MALDI MSI workflow capable
of achieving a spatial resolution of 250 to 300 nanometers, comparable to the level of
optical microscopy, for detailed subcellular molecular visualization. The core idea of this
research is to enhance the spatial resolution of MALDI mass spectrometry imaging by
physically expanding the sample size. We originally proposed and developed the concept
and method of Expansion Mass Spectrometry Imaging (ExMSI), which overcomes the
limitations and challenges of hardware design for ultra-small laser spots and sample
preparation. In previous work, we successfully reached 500 nm resolution with a 10-fold
sample expansion. Now, we aim to push the spatial resolution further, to 250 to 300 nm,
using a 20-fold expansion.
This advancement will enable imaging at the nanometer scale with the ability to detect a
wide range of molecules while preserving their integrity. The proposed ExMSI platform
will provide an unprecedented view of molecular details at the subcellular level in
cultured cells and brain tissues, significantly advancing our understanding in fields like
cellular biology and neuroscience. It represents a powerful tool for researchers seeking
new insights into the complex molecular processes underlying health and disease.