Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

Abstract

Summary Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small molecule probes or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra (EELS) of each lanthanide deposit via energy-filtered transmission electron microscopy (EFTEM). This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the post-synaptic membrane.

Multi-color electron microscopy by element-guided identification of cells, organelles and molecules

“…EDX spectroscopy and imaging on cryo-fixed tissue has been pioneered by Somlyo and coworkers8910, and pioneering studies have mainly focused on detection of a few selected elements in small regions at relatively low resolution (see for example1112). Leapman and co-workers applied and pioneered electron energy loss spectroscopy (EELS) in transmission EM to discriminate cells based on sequential analysis of three elements13, and recently Tsien and coworkers presented EELS-based two-color discrimination of localized deposits of lanthanides14. EDX allows direct identification of many elements in parallel, either present endogenously and/or introduced by staining or labeling, at high count rates using the latest generation of silicon drift detectors (SDD).…”

Application of EELS and EFTEM to the life sciences enabled by the contributions of Ondrej Krivanek

“…In 2008, Roger Tsien won the Chemistry Nobel prize (together with Osamu Shimomura and Martin Chalfie) for developing genetically encoded green fluorescent protein (GPF), which has revolutionized studies of the biology of living cells in the optical microscope, and the rare earth EFTEM probes were conceived as a way to extend the GFP labeling approach to a finer scale in the electron microscope. In this recent work, the University of California San Diego team developed a genetically encoded photoactivated, singlet oxygen generator that, on activation, polymerizes the compound diaminobenzidine (DAB) that is chelated with a rare earth element [40]. After sample preparation for electron microscopy, the rare earth atoms are retained in close proximity to the location of the original genetic tag.…”

“…After sample preparation for electron microscopy, the rare earth atoms are retained in close proximity to the location of the original genetic tag. In their recent paper published in Cell Chemical Biology, Adams et al genetically tagged the complementary DNA for an important brain protein PKM-ζ involved in long-term potentiation, i.e., memory, with DNA for fluorescent protein and a singlet oxygen generator [40]. In these experiments, transfected cultured rat neurons were stimulated with a compound to induce long-term potentiation, followed by addition of cerium-chelated DAB, then photoactivation to generate singlet oxygen species and hence DAB polymerization, and finally the specimens were prepared for electron microscopy.…”

“…In their experiments, Adams et al used an in-column Omega filter on a JEOL JEM-3200EF TEM and an Ultrascan 4000 CCD detector or a Direct Electron DE-12 detector. They found cerium deposited at the post-synaptic membrane in stimulated neurons but not in the controls, demonstrating that PKM-ζ had indeed been produced following stimulation [40]. This result suggests that EFTEM could play an important role in mapping specific proteins in cells and tissues on the scale of a few tens of nanometers rather than the approximately ten times large scale of optical fluorescence microscopy.…”

Boron‐Containing Probes for Non‐optical High‐Resolution Imaging of Biological Samples

“…One could in principle,u se goldlabeled antibodies as in EM. [13,14] As imple and flexible solution to this could be to use boron, which typically has as trong signal in techniques such as nanoSIMS or EELS. [10][11][12] At the same time,ithas been noted that gold particles appear to modify the sample ionization in their surroundings,f or example for CN À ions.…”