Multi-colour electron microscopy delivered
Image: Two-colour electron microscopy image of endosomal uptake of peptide proteins. [Adams et al./Cell Chemical Biology 2016]
In a world first, US-based researchers have unveiled multi-colour electron microscopy images captured via electron energy loss spectroscopy and energy-filtered TEM.
The two-colour images of cellular membranes and synaptic connections between brain cells follow nearly fifteen years of development from Chemistry Nobel Prize laureate, Professor Roger Tsien and his team. Tsien died suddenly earlier this year.
Two-colour merge of the spectrally separated elemental maps (green for Ce and red for Pr) overlaid on a conventional image, showing the two different astrocyte processes contacting the same synapse. [Adams et al./Cell Chemical Biology 2016]
"One theme that has gone through all of Roger's work is the desire to peer more closely into the workings of the cell," highlights University of San Diego chemist, Stephen Adams.
"With all of the fluorescence techniques that he's introduced, he was able to do that in live cells, and make action movies of them in vivid colours," he adds. "But he always wanted to look closer, and now he's left the beginnings for a method where we can add colours to electron microscopy."
With the new method, up to three colours at a time - green, red and yellow - can be used in an image.
To obtain the multi-colour effect, the researchers coat specimens with metal complexes - ionised lanthanun, cerium and praseodymium - that can withstand application and have a distinct eletron enery loss signature.
During analysis, the energy detector on the JEOL JEM-3200EF TEM captures the electrons lost from metal ions deposited over the samples, recording the energy loss signature as a colour.
A detailed view of biological structures is then created by overlaying conventional electron micrographs with the pseudocolour lanthanide elemental maps derived from the electron energy loss spectra.
With the application process in place, the researchers went onto visualise two brain cells sharing a single synapse, and also showed peptides entering a cell membrane.
Two-colour merge of the elemental maps (La in green and Ce in red), overlaid on the conventional electron microscopy image of Golgi and Plasma Membrane in Tissue Culture Cells. [Adams et al./Cell Chemical Biology 2016]
According to the researchers, the method is analogous to fluorescence microscopy but provides the full spatial resolution of electron microscopy.
"It's a bit like when you first see a colour photograph after having only known black and white; for the last 50 years or so, we've been so used to monochrome electron micrographs that it's now hard to imagine that we could go back," says Adams. "This method has many potential applications in biology; we demonstrate how it can distinguish cellular compartments or track proteins and tag cells."
But despite the breakthrough, the sample preparation method is complex and the researchers admit more chemistry is needed to perfect the metal ion application process as well as produce images with more than three colours.
However, they believe the biochemical community should be able to start to use the technique using existing laboratory tools.
"This is clearly an example of Roger's brilliance at chemistry and how he saw that if we could do this, we would be able to enjoy the advantages of electron microscopy," says Dr Mark Ellisman, director of the National Center for Microscopy and Imaging Research at the University of California, San Diego.
"The biggest advantage of electron microscopy that we saw is that you have weak contrasts by the nature of the way that staining works so colour-specific labels give context to all of the rich information in the scene of which molecules are operating," he adds.
Research is published in Cell Chemical Biology.