Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Molecular Imaging

Background:

• Electron microscopy (EM) is crucial for ultrastructural imaging but struggles with simultaneous multi-molecule localization.
• Current EM methods lack the multiplexing capability seen in fluorescence imaging.
• Precisely mapping multiple subcellular components within their native context remains a significant challenge.

Purpose of the Study:

• To develop a novel multicolor electron microscopy method for simultaneous localization of multiple subcellular components.
• To achieve multicolor EM imaging with the full spatial resolution of conventional EM.
• To demonstrate the utility of this technique in visualizing complex biological structures and molecular distributions.

Main Methods:

• Sequential, localized deposition of different lanthanides using photosensitizers, small-molecule probes, or peroxidases.
• Overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps.
• Utilizing distinctive electron energy-loss spectra and energy-filtered transmission electron microscopy for lanthanide mapping.

Main Results:

• Generation of multicolor EM images with high spatial resolution, analogous to multicolor fluorescence.
• Visualization of hippocampal astrocytes revealing shared synapses between adjacent astrocyte processes.
• Demonstration of polyarginine-based cell-penetrating peptide uptake via endocytosis.
• Observation of preferential postsynaptic membrane localization of newly synthesized PKMζ in cultured neurons.

Conclusions:

• The developed multicolor EM method overcomes limitations in simultaneous molecular localization.
• This technique provides unprecedented insights into subcellular organization and molecular interactions.
• The methodology has broad applications in cell biology, neuroscience, and molecular imaging research.