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New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast
I Cornelese-ten Velde1, F A Prins
1Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands.
Insights
A new post-embedding method combines light and electron microscopy for antigen detection. Reflection contrast microscopy (RCM) enhances immunogold staining sensitivity compared to bright-field microscopy (BFM).
Area of Science:
- Immunohistochemistry
- Microscopy techniques
- Pathology
Background:
- Combined light and electron microscopy (CLEM) is crucial for detailed cellular and subcellular analysis.
- Traditional immunogold labeling methods can face limitations in sensitivity and visualization across different microscopy platforms.
- Lowicryl K4M embedding offers good preservation for CLEM applications.
Purpose of the Study:
- To present a simple post-embedding method for CLEM.
- To evaluate the potential of reflection contrast microscopy (RCM) for visualizing immunogold-labeled antigens.
- To compare the sensitivity of RCM with bright-field microscopy (BFM) for immunogold detection.
Main Methods:
- Immunogold (silver) staining was performed on Lowicryl K4M-embedded rat and mouse kidney tissues, including those with experimental nephritis.
- Labeling was visualized using bright-field microscopy (BFM), reflection contrast microscopy (RCM), and electron microscopy.
- The study focused on the detection of colloidal gold particles (15 nm) and silver-enhanced gold particles on ultrathin sections.
Main Results:
- Successful detection of immunogold and silver-enhanced gold particles was achieved on ultrathin sections using RCM.
- RCM demonstrated increased detection sensitivity compared to BFM for visualizing gold particles.
- RCM visualization enhanced the sensitivity of the immunostaining procedure.
Conclusions:
- The presented post-embedding method enables effective CLEM for antigen detection.
- Reflection contrast microscopy is a valuable tool for enhanced visualization and sensitivity in immunogold labeling.
- RCM offers advantages over BFM for detecting fine gold particles in various microscopy applications.
Abstract:
One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.
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