Multi-Color Electron Microscopy by Element-Guided Identification of Cells, Organelles, and Molecules

Abstract

Electron microscopy (EM) is a powerful tool to study cellular complexity at nanometer resolution. Traditionally, EM images are acquired from small fields of view, but several new techniques allow routine acquisition of large 2D and 3D image stacks. However, in the resulting vast amount of greyscale data interpretation in terms of macromolecular functionality is difficult. With Correlative Light and Electron Microscopy (CLEM), fluorescence can be used to identify molecules in color[1] but the resolution gap with EM precludes accurate localization. The use of superresolution fluorescence microscopy techniques combined with CLEM[2,3] holds great promise but may be hard to achieve and the color palette limited. We are exploring novel approaches to achieve color-EM using the electron beam to add information about the specimen to the electron micrographs.Large-scale EM is complemented with energy-dispersive-X-ray analysis (EDX) to allow EM analysis based on elemental composition. Different endogenous elements as well as exogenously introduced elements, including labels (gold-and cadmium-based nanoparticles) and stains, can be discriminated at ultrastructure resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exists in single cells in a Type-1 diabetes rat model (see Figure 1), highlighting how elemental mapping provides biomedical relevant information without bias. Broad application of EM-EDX will further allow experimental analysis on different kind of tissues, stains, and labels and thus brings nanometer-scale 'color-EM' as a promising tool to unravel molecular (de)regulation in biomedicine.

State‐of‐the‐art microscopy to understand islets of Langerhans: what to expect next?

“…While for light microscopy immunolabeling is very straightforward, immunolabeling on EM samples is very limited because of conflicting sample preparation requirements. However, protocol optimization has enabled immuno‐EM labeling of insulin, 32,33 glucagon 34 and somatostatin 35 for correlative light and EM, which allows fluorescence guidance of gray‐scaled EM samples. More recently, elemental analysis is becoming a routine application in the life sciences, helping identify structures in EM micrographs.…”

“…In addition to the analysis of electrons, now the loss of electron energy 36 or energy of emitted X‐rays from the sample is also determined (Figure 3c–e). 34 Thus, adding multimodality helps to identify molecules or structures in EM maps, that is, the differentiation between granules or cell types 12 is guided by adding information from the additional detector typically in false color, and therefore popularly called “ColorEM” (Figure 3c–e; Table 1). 37 ColorEM data mining of the nPOD nanotomy repository for zoomable EM maps 12 revealed an increased cellular colocalization of both exocrine and endocrine secretory granules in autoantibody‐positive and T1D donor samples accompanied by aberrant ultrastructural morphology, which might indicate an exocrine pancreatic involvement in T1D etiopathology.…”

Elemental Mapping of Labeled Biological Specimens at Intermediate Energy Loss in an Energy-Filtered TEM acquired using a Direct Detection Device

“…Although EFTEM mode has lower sensitivity than STEM-EELS, it has at least an order of magnitude larger field of view, typically 10 5 – 10 7 pixels in comparison to 10 3 – 10 5 pixels in STEM-EELS (Aronova & Leapman, 2012; Leapman, 2017). For certain biological applications, a larger field of view is just as important as either resolution or sensitivity, as is the case for the application of multicolor electron probes to simultaneously label multiple cellular proteins/organelles in cells (Adams, et al, 2016; Pirozzi, et al, 2018; Scotuzzi, et al, 2017). In the method that we developed, the localization of the multiple targeted molecules was achieved by the sequential deposition of specific lanthanide chelates conjugated to diaminobenzidine, which were selectively oxidized by orthogonal photosensitizers / peroxidases (Adams, et al, 2016).…”

SEM, XRD and EDX analysis of calcium mineralization in human aortic valve

“…Energy dispersive X-ray (EDX) spectroscopy is an analytical technique used to obtain elemental analysis or chemical combination of a sample and it is usually integrated into SEM. It is also used to obtain quantitative elemental analysis and 2-dimensional elemental mappings of different compositions (Scotuzzi et al, 2017;Goldstein et al, 2017). In the last decade, EDX-integrated SEM has been used to rapidly and accurately demonstrate the morphology of biological samples and the chemical composition of structures in this morphology (Delogne et al, 2007;Jung et al, 2015;Li et al, 2017;Scimeca et al, 2018;Perrotta and Davoli, 2014).…”