Abstract
A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.
Highlights
Imaging of human and animal tissues, individual cells, and subcellular structures plays a key role in biomedicine and cell biology research
MSI and scanning electron microscopy (SEM) closer than ever before, we report a procedure for the processing of frozen sections for SEM from lung specimens initially collected and stored in a deep-frozen state for MALDI-MSI
After the thaw-mounting step, the surface of the vacuum-dried sections was covered with a distinct layer of dried colloids of intra- and extracellular origin, which did not interfere with the MALDI-MSI analysis but made
Summary
Imaging of human and animal tissues, individual cells, and subcellular structures plays a key role in biomedicine and cell biology research. Merging visual morphology information with molecular or elemental mass spectrometry data is another challenge. J. Fungi 2020, 6, 257 imaging (MSI) data fusion. From a mass spectrometry point of view, the most common MALDI-MSI method allows for label-free mapping of molecules, including proteins, peptides, lipids, metabolites, and small molecules, directly in tissue. The physicochemical information in a single spot may involve the molecular formula, relative ion abundance, and organic structure-specific product ion mass spectrum. To reveal structure- and function-specific information, it is possible to combine MALDI-MSI molecular maps of tissue sections with classical histology [3,4] and fluorescence microscopy [5,6]
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