Correlative Microscopy Combining Secondary Ion Mass Spectrometry and Electron Microscopy: Comparison of Intensity–Hue–Saturation and Laplacian Pyramid Methods for Image Fusion

Correlative microscopy combining various imaging modalities offers powerful insights into obtaining a comprehensive understanding of physical, chemical, and biological phenomena. In this article, we investigate two approaches for image fusion in the context of combining the inherently lower-resolution chemical images obtained using secondary ion mass spectrometry (SIMS) with the high-resolution ultrastructural images obtained using electron microscopy (EM). We evaluate the image fusion methods with three different case studies selected to broadly represent the typical samples in life science research: (i) histology (unlabeled tissue), (ii) nanotoxicology, and (iii) metabolism (isotopically labeled tissue). We show that the intensity–hue–saturation fusion method often applied for EM-sharpening can result in serious image artifacts, especially in cases where different contrast mechanisms interplay. Here, we introduce and demonstrate Laplacian pyramid fusion as a powerful and more robust alternative method for image fusion. Both physical and technical aspects of correlative image overlay and image fusion specific to SIMS-based correlative microscopy are discussed in detail alongside the advantages, limitations, and the potential artifacts. Quantitative metrics to evaluate the results of image fusion are also discussed

Additional file 1: Figure S1. of Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium

Mucus layer characterization (Alcian blue staining). Figure S2. Mucus layer characterization (Toluidine blue staining and TEM). Figure S3. Mucus layer characterization (Toluidine blue staining, top view). Figure S4. Cell monolayer integrity evaluation (TEER). Figure S5. Cell-free DCFH-DA assay. Figure S6. TEM images of cells in co-culture exposed to Ag particles. Figure S7. Hierarchical clustering. Table S1. Detailed information on protein identification. Table S2. Cellular Ag content determination. Table S3. KEGG enrichment analysis. (DOCX 1.17 mb