Simultaneous detection of multiple targets for ultrastructural immunocytochemistry

Abstract

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

“…A variant on this approach has been the use of colloidal gold and quantum dot particles in combination with scanning transmission electron microscopy (STEM) and elemental composition analysis (Loukanov et al 2010). An alternative approach (Philimonenko et al 2014) has utilised other electron-dense nanoparticles identifiable on the basis of their differences in shape rather than size. With this approach, five different targets have been localised simultaneously in the nuclei of Hela cells by using two different sizes of colloidal gold (6 nm and 12 nm) together with gold-silver core-shell nanoparticles (doughnut-shaped, ∼13 nm), palladium nanoparticles (cubic, ∼15 nm) and gold nanoparticles (rod-shaped, ∼16 nm×6 nm).…”

“…Detection and localisation depend on the target specificity of the primary antibody and the ability of TEM to resolve the various compartments and visualisation markers. The ability to identify and quantify the readout is facilitated by the availability of visualisation markers that are electron-dense and point-like and that differ in size and/or shape (see Griffiths 1993;Philimonenko et al 2014). For instance, colloidal gold particles are available in various sizes (usually 5-20 nm).…”

Morphomics: An integral part of systems biology of the human placenta

“…This requires the use of more than one size and/or shape of probe particle, each associated with a different target [133,137].…”

Colocalization coefficients evaluating the distribution of molecular targets in microscopy methods based on pointed patterns

“…In such a way, the target molecules become visible due to the colloidal metal particles which are conjugated with the antibodies. The different types of particles of the various size, shape, and material can substitute the different colors of fluorochromes used for labeling in light microscopy (Philimonenko et al 2014 ).…”