Related Experiment Videos
Imaging of cytoskeletal elements by low-temperature high-resolution scanning electron microscopy
Y Chen1, V E Centonze, A Verkhovsky
1Integrated Microscopy Resource (IMR), University of Wisconsin-Madison 53706, USA.
Journal of Microscopy
|July 1, 1995
Summary
High-resolution scanning electron microscopy (HRSEM) visualized actin filaments and microtubules in 3D. This cryo-technique revealed detailed cytoskeletal architecture and subunit structures with high contrast and resolution.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biophysics
Background:
- Cytoskeletal elements like actin filaments and microtubules are crucial for cellular structure and function.
- Previous imaging techniques had limitations in visualizing the intricate 3D architecture of the cytoskeleton with high resolution.
Purpose of the Study:
- To develop and apply a low-temperature high-resolution scanning electron microscopy (HRSEM) technique for detailed imaging of cytoskeletal structures.
- To examine the three-dimensional architecture and structural relationships of actin filaments and microtubules in situ and in vitro.
Main Methods:
- Specimens (fibroblasts and purified proteins) were prepared, fixed, and subjected to plunge freezing.
- Controlled freeze-drying, cryo-sputter coating with chromium, cryo-transfer, and cryo-observation in a Field Emission Scanning Electron Microscope (FESEM) were employed.
- High-resolution scanning electron microscopy (HRSEM) was utilized for imaging.
Main Results:
- The cryo-HRSEM technique successfully preserved the three-dimensional architecture of the cytoskeleton.
- Actin filaments exhibited characteristic helical features with revealed periodicities (5.5 nm and 37 nm).
- Microtubules showed individual protofilaments and a 4-nm repeat of tubulin subunits; clathrin cages were also observed.
Conclusions:
- The developed cryo-HRSEM method offers a powerful approach for direct imaging of macromolecular structures.
- This technique provides high contrast and signal-to-noise ratio at a resolution of 2-3 nm, enabling detailed examination of cytoskeletal organization.