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A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Published on: March 30, 2020
Simultaneous detection of multiple targets for ultrastructural immunocytochemistry
V V Philimonenko1, A A Philimonenko, I Šloufová
1Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14200, Prague 4, Czech Republic.
Insights
Researchers developed novel metal nanoparticles for electron microscopy, enabling simultaneous detection of up to five molecular targets. This breakthrough in ultrastructural histochemistry enhances the study of cellular localization and molecular interactions.
Area of Science:
- Cell Biology
- Nanotechnology
- Biochemistry
Background:
- Indirect immunolabeling is crucial for understanding molecular localization and interactions within cells.
- Fluorescent microscopy allows multiplexed antigen detection, but electron microscopy (EM) is limited to two targets.
- Existing EM immunodetection methods lack the capacity for simultaneous analysis of multiple molecules.
Purpose of the Study:
- To overcome the limitation of simultaneous antigen detection in electron microscopy.
- To develop novel, shape-coded metal nanoparticles for enhanced EM immunodetection.
- To enable the simultaneous identification of up to five molecular targets in ultrastructural histochemistry.
Main Methods:
- Conjugation of novel, shape-coded metal nanoparticles to antibodies or bioreactive molecules.
- Combination of these novel nanoparticles with commercial gold nanoparticles.
- Application in transmission electron microscopy for ultrastructural analysis.
Main Results:
- Successful preparation of novel, shape-coded metal nanoparticles.
- Demonstration of simultaneous detection of up to five molecular targets using these nanoparticles.
- Mapping of nuclear lipid phosphatidylinositol-4,5-bisphosphate and four other genome-related molecules.
Conclusions:
- The developed nanoparticles significantly advance EM-based multiplexed molecular detection.
- This method allows for unprecedented simultaneous analysis of multiple targets at the ultrastructural level.
- The technique holds broad potential for diverse biomedical applications, particularly in cell biology and molecular mapping.
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.
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