Integration of a high-NA light microscope in a scanning electron microscope

Area of Science:

• Correlative microscopy
• Nanotechnology
• Materials Science

Background:

• Simultaneous imaging with light and electron microscopy is challenging.
• Existing methods often lack precise alignment and high resolution.

Purpose of the Study:

• To present an integrated light-electron microscope for simultaneous high-resolution imaging.
• To enable correlative light and electron microscopy on the same sample area.

Main Methods:

• An inverted high-numerical aperture (NA) objective lens was integrated inside a scanning electron microscope (SEM).
• The light and SEM objective lenses share a common axis and focal plane.
• In situ epifluorescence microscopy with a NA of 1.4 was performed using vacuum-compatible immersion oil.

Main Results:

• Simultaneous high-resolution optical and scanning electron microscopy was achieved.
• Precise alignment of fields of view was demonstrated (sub-10 μm).
• A 40-nm fluorescent nanoparticle showed a full width at half maximum (FWHM) of 380 nm, with uncompromised SEM performance.

Conclusions:

• The integrated instrument offers new possibilities for correlative light and electron microscopy.
• This technique is applicable to life sciences, physics, and chemistry.
• The system provides flexibility in light microscope component mounting.