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Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes
Published on: March 20, 2018
Labeling of ultrathin resin sections for correlative light and electron microscopy
Gunar Fabig1, Susanne Kretschmar, Susanne Weiche
1Center for Regenerative Therapies, TU Dresden, Fetscherstraße 105, D-01307 Dresden, Saxony, Germany.
Insights
This study presents fast methods for correlative immunofluorescence and immunogold labeling on ultrathin sections. These techniques enable direct correlation of fluorescent signals with subcellular structures using light and electron microscopy.
Area of Science:
- Cell Biology
- Microscopy Techniques
Background:
- Correlative microscopy integrates light and electron microscopy for enhanced spatial resolution.
- Simultaneous labeling is crucial for accurate correlation of biological structures.
Purpose of the Study:
- To develop fast and simple methods for correlative immunofluorescence and immunogold labeling on ultrathin sections.
- To enable direct correlation of fluorescent signals with subcellular structures.
Main Methods:
- Demonstrated on methacrylate (Lowicryl K4M) embedded tissue sections.
- Simultaneous staining with fluorescent and gold markers (Protein A gold, IgG gold, Alexa dyes, FluoroNanogold) on EM grids.
- Sequential analysis using light microscopy and transmission electron microscopy (TEM).
Main Results:
- Successful correlative immunofluorescence and immunogold labeling achieved.
- Fluorescent signals were directly correlated with specific subcellular structures in TEM.
- Demonstrated the utility of various gold and fluorescent markers, including silver enhancement.
Conclusions:
- The developed protocols are fast, simple, and effective for correlative imaging.
- This method allows high-resolution analysis of fluorescently labeled structures identified by light microscopy.
- Enables precise localization of targets within subcellular compartments.
Abstract:
Correlative microscopy combines the versatility of the light microscope with the excellent spatial resolution of the electron microscope. Here, we describe fast and simple methods for correlative immunofluorescence and immunogold labeling on the very same ultrathin section. The protocols are demonstrated on sections of tissue samples embedded in the methacrylate Lowicryl K4M. Ultrathin sections are mounted on electron microscopy (EM) grids and stained simultaneously with fluorescent and gold markers. For the detection of primary antibodies, we applied either protein A gold or immunoglobulin G (IgG) gold in combination with secondary antibodies coupled to Alexa488 or Alexa555. Alternatively, the correlative marker FluoroNanogold was used, followed by silver enhancement. The samples have to be analyzed first at the light microscope and then in the transmission electron microscope (TEM), because the fluorescence is bleached by the electron beam. Labeled structures selected at the fluorescence microscope can be identified in the TEM and analyzed at high resolution. This way, fluorescent signals can be directly correlated to the corresponding subcellular structures in the area of interest.
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