2021-01-21_José María Mateos_3D CLEM Based On Non-Contrasted Tissue Sections

3D CLEM Based On Non-Contrasted Tissue Sections

The talk presents a research project focused on developing a method for 3D correlative light and electron microscopy (CLEM) using non-contrasted tissue sections. Jose Maria Mateos discusses the historical development, technical challenges, and advantages of this approach. The method leverages Tokuyasu cryo-sectioning techniques, silicon wafers for stability, and low-voltage scanning electron microscopy (SEM) to achieve high-resolution imaging without heavy metal contrasting. Key insights include the method's effectiveness in 2D imaging, its potential for super-resolution techniques like SURF, and the challenges encountered in extending it to 3D applications.

Main research question

How can correlative light and electron microscopy be optimized for high-resolution imaging of biological samples without the use of heavy metal contrasting, and what are the challenges and advantages of extending this method to 3D?

Presenter

José María Mateos

Key insights from the talk

• The method combines light microscopy and electron microscopy to provide both protein localization and tissue context, addressing limitations of each technique individually.
• Non-contrasted tissue sections imaged at low voltage in SEM yield high-resolution details without the need for heavy metal staining, preserving fine biological structures.
• Silicon wafers provide stability and flatness for imaging, making the method robust and easier to handle compared to traditional EM grids.
• Super-resolution techniques like SURF (Super-Resolution Optical Fluctuation Imaging) can be effectively integrated with this method, offering faster imaging and improved resolution for thin sections.
• While the method excels in 2D imaging, extending it to 3D presents challenges such as physical distortions in serial sections and difficulties in maintaining sample orientation.
• The use of glycerol during light microscopy imaging prevents sections from drying out, ensuring sample integrity.
• The method allows for multi-protein labeling and large field-of-view imaging, making it versatile for various biological applications.