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 Secondary Ion Mass Spectrometry and Electron Microscopy: Comparison of Intensity–Hue–Saturation and Laplacian Pyramid Methods for Image Fusion
Vollnhals F, Audinot J-N, Wirtz, T, Mercier-Bonin M, Fourquaux I, Schroeppel B, Kraushaar U, Lev-Ram V, Ellisman MH, Eswara S
Anal. Chem. (2017), Bd. 89, H. 20, S. 10702-10710, https://doi.org/10.1021/acs.analchem.7b01256

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.