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Multicolor electron microscopy paints multiple cellular structures

By Melissae Fellet February 7, 2017

Biologists want to see the locations within cells of proteins, membranes, and cellular structures like the nucleus, when exploring the inner workings of cells. Electron microscopy is able to provide images of these structures at atomic resolution, but it is hard to label specific components to uniquely identify them. One common staining method for electron microscopy labels a structure with an antibody carrying a fluorescent dye. When excited by light, these dyes generate reactive oxygen species that induce precipitation of a small molecule, diaminobenzidine (DAB). Adding osmium subsequently makes the DAB precipitate visible in transmission electron microscopy, but a DAB-coated structure may be hard to distinguish from other cellular components that also attract osmium.

Two cell structures, surrounded by red or green circles to represent fluorescent dyes, are sequentially coated with different lanthanide ions chelated to a diaminobenzidine (DAB) ligand. Researchers infuse the cell with osmium, which stains the introduced DAB as well as other cellular structures. Finally, energy filtered transmission electron microscopy (EF-TEM) gathers images of cell structures stained by the osmium and each lanthanide. The three images are combined to visualize each labeled structure in relationship to each other and the other cellular contents. Credit: Cell Chemical Biology

As recently reported in Cell Chemical Biology, Stephen Adams at the University of California, San Diego, and his colleaguesave developed DAB conjugates carrying different lanthanide ions, enabling them to obtain electron micrographs of cells with labels on two different structures. Each lanthanide ion has a unique signature in electron energy loss spectroscopy as implemented by energy-filtered transmission electron microscopy (EELS-EFTEM), a method typically used to study the surface of materials. Using this new labeling approach, the researchers imaged the borders of two different brain slices, as well as a protein localizing in an area that is too electron dense to image with traditional electron microscopy.

Sample preparation to visualize multiple cellular structures is similar to that used for the current DAB staining. The researchers start with a cell containing two different fluorescent labels. They then introduce one lanthanide-DAB conjugate, followed by light-induced precipitation of the DAB, to coat one of the labeled cell structures with the lanthanide ion. Next, they repeat the DAB precipitation using a different lanthanide-DAB conjugate. Finally, the researchers infuse the cell with osmium.

To image both labeled cellular structures, the researchers first collect a transmission electron micrograph of the osmium-labeled structures. Next, they use EELS-EFTEM to visualize the signatures from each lanthanide-labeled structure. Finally, the researchers overlay each EELS image onto the osmium TEM image, providing a picture of the labeled structures in relation to each other and the rest of the cell.

Using this technique, the researchers were able to visualize projections from two different astrocytes, non-neuronal cells in the brain, contacting the same synapse. They also imaged a newly-synthesized protein gathering on one side of the junction between nerve cells, an area too electron-rich to get good resolution with traditional EM.

The goal of this technique is to bridge fluorescence microscopy and electron microscopy, says Stephen Adams, at the University of California, San Diego. With fluorescence microscopy, researchers can follow the dynamic metabolism inside a live cell. Chemically rigidifying a cell for electron microscopy freezes the activity and provides a detailed snapshot of the locations of labeled biomolecules.

Richard Edelmann, at Miami University, thinks this lanthanide staining would be easy for microscopists to use because it is a variation of current DAB staining procedures. But he wonders if this method will find wide applicability because few institutions have the expensive microscopes needed to detect the weak lanthanide signal.

Adams says they are working on increasing the signal by introducing more lanthanide ions.

Read the abstract in Cell Chemical Biology.