Next Generation Histology-Directed Imaging Mass Spectrometry Driven by Autofluorescence Microscopy

Area of Science:

• Biomedical Imaging
• Mass Spectrometry
• Molecular Pathology

Background:

• Histology-directed imaging mass spectrometry (IMS) traditionally requires serial tissue sections for annotation and analysis.
• Staining methods for annotation are often incompatible with IMS, complicating spatial targeting.
• Accurate registration between annotated and IMS sections is crucial but challenging.

Purpose of the Study:

• To develop a next-generation histology-directed platform for IMS using autofluorescence (AF) microscopy.
• To enable streamlined, spatially targeted molecular characterization of tissues.
• To demonstrate the platform's utility in disease-specific case studies.

Main Methods:

• Implementation of IMS-compatible autofluorescence (AF) microscopy prior to staining or IMS acquisition.
• Development of two histology-directed workflows: automated AF-to-AF image registration and a registration-free direct AF targeting approach.
• Application of the platform to liver-stage malaria and human kidney disease research.

Main Results:

• Automated AF-to-AF registration achieved high accuracy (∼3-10 μm) for annotation transfer across multiple organs and species.
• The registration-free AF approach allowed direct targeting of fine structures visible via AF microscopy.
• Successful spatially targeted IMS acquisition of rare malaria-infected hepatocytes and kidney glomeruli was demonstrated.

Conclusions:

• The novel platform enhances histology-directed IMS by integrating AF microscopy.
• It offers both improved registration accuracy and a registration-free option for precise spatial targeting.
• This technology advances molecular pathology research by enabling efficient analysis of specific, rare cellular targets in disease contexts.