title: Integration of a high-NA light microscope in a scanning electron microscope.
abstract: We present an integrated light-electron microscope in which an inverted high-NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub-10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum-compatible immersion oil. For a 40-nm-diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.
authors.affiliation: Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg, the Netherlands.
authors.forename: A C
authors.initials: AC
authors.lastname: Zonnevylle
authors.forename: R F C
authors.initials: RF
authors.lastname: Van Tol
authors.forename: N
authors.initials: N
authors.lastname: Liv
authors.forename: A C
authors.initials: AC
authors.lastname: Narvaez
authors.forename: A P J
authors.initials: AP
authors.lastname: Effting
authors.forename: P
authors.initials: P
authors.lastname: Kruit
authors.forename: J P
authors.initials: JP
authors.lastname: Hoogenboom
mesh_terms: D002460:Cell Line; D031202:Chrysanthemum; D004847:Epithelial Cells / Q000166:cytology / Q000648:ultrastructure; D006801:Humans; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D011058:Pollen / Q000166:cytology / Q000648:ultrastructure
keywords: Cathodoluminescence microscopy; cellular imaging; correlative microscopy; fluorescence microscopy; light microscopy; scanning electron microscopy