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.

Department of Analytical Chemistry Faculty of Science Palacký University 17 listopadu 12 771 46 Olomouc Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 128 44 Prague Czech Republic

Faculty of Science J E Purkyně University České mládeže 8 400 96 Ústí nad Labem Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Hněvotínská 5 779 00 Olomouc Czech Republic

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