Thesis Defense: Yunhao Bai, Nolan Group

"Adapting Expansion Microscopy to Imaging Mass Spectrometry"

The recent development of Expansion Microscopy (ExM) and related techniques physically enlarge samples for enhanced spatial resolution while largely retaining native biomolecular content and coordinates, but almost exclusive to fluorescent imaging as the readout modality. On the other hand, mass spectrometry imaging (MSI) has been used to acquire spatial multiplex or omics information and excels at interrogating cellular positioning in tissues but is currently limited in the ability to resolve biomolecular features at high-resolution, while instrumental modification is costly prohibited and limited by physics. Combining ExM with tissue MSI technologies would enable integrative multiscale tissue biology studies. This ostensibly straightforward idea is counterintuitive, as expanded ExM gel is similar to a sponge full of water, while most of MSI methodologies require water-free to achieve decent resolution.

This thesis will introduce an ExM framework that not only allows the total removal of water inside hydrogels while retaining its lateral magnification, but also preserves the biomolecules inside expanded archival clinical samples. Processed gel can be integrated into existing tissue staining protocols and instrumentation without much modification. This framework allows the multiplexed MSI of proteomics by tagged antibodies with resolution gain as the expansion fold, and it has been applied to archival human lymphoid and brain tissues to resolve orchestrated features of tissue architecture, particularly that of the blood-brain barrier (BBB). Besides this, multiplex imaging of N-glycan that previously inaccessible by fluorescence imaging in expanded archival tissues can be acquired through MALDI-TOF, to push it towards the unpreceded single-cell resolution.