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Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM)
Yuusuke Maruyama1, Tatsuhiko Ebihara, Hidetoshi Nishiyama
1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki, Japan.
Journal of Structural Biology
|September 11, 2012
Summary
The atmospheric scanning electron microscope (ASEM) enables correlative optical and scanning electron microscopy of wet cell cultures. This technique visualizes cellular structures like cytoskeletons and protein dynamics in real-time.
Area of Science:
- Cell Biology
- Microscopy
- Biophysics
Background:
- Observing dynamic cellular processes in wet environments requires advanced microscopy techniques.
- Traditional electron microscopy methods often necessitate sample fixation, limiting in situ observation of live cells.
- Correlative microscopy combines the strengths of different imaging modalities for comprehensive analysis.
Purpose of the Study:
- To explore the utility of atmospheric scanning electron microscopy (ASEM) for in situ correlative optical/scanning electron microscopy (OM/SEM) immuno-microscopy.
- To assess the efficiency of dual-tagged labeling for correlative imaging.
- To document the imaging capabilities of the ASEM system for live cell analysis.
Main Methods:
- Utilized an inverted scanning electron microscope (SEM) observing samples from beneath a disposable dish with a silicon nitride (SiN) film window.
- Employed an optical microscope (OM) for simultaneous top-down observation of cultured cells.
- Visualized cytoskeletal components (actin, tubulin), endoplasmic reticulum (ER) chaperone PDI, and calcium sensor STIM1 using established cell lines and primary neuron cultures.
Main Results:
- Successfully visualized cytoskeletons, PDI in the ER, and STIM1 in ER membranes.
- Observed dynamic STIM1 aggregation in Jurkat T cells upon calcium depletion and detailed actin and tubulin organization in neuronal growth cones and synapses.
- Demonstrated partial colocalization of actin fibers with Homer1c in neuronal lamellipodia and distinct F-actin arrangements in post-synaptic structures.
Conclusions:
- The ASEM system provides a viable platform for in situ correlative OM/SEM immuno-microscopy of wet cell cultures.
- The technique allows for high-resolution visualization of cellular structures and protein localization in living cells.
- ASEM correlative microscopy holds significant promise for investigating the dynamics of protein complexes and cellular events in the near future.
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