Abstract
Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.
Highlights
For three-dimensional (3D) ultrastructural investigations with electron microscopy (EM), five different techniques are typically used (Fig. 1)
We focused on the following aims: (i) to design and produce slides and coverslips with a variety of customized coordinates for correlative light microscopy (LM) and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM of both critical point dried (CPD) and flat embedded samples; (ii) to establish a labeling technique for “post-embedding”; (iii) to evaluate strategies to immobilize cells and tissue sections; (iv) to develop a filter system for “flat embedding” of large, fragile or delicate specimens; and (v) to use thick epoxy sections for highresolution LM, transmission EM (TEM), and FIB/SEM tomography
We first tested several methods to label coordinates on glass slides and coverslips. All these labels were clearly visible in LM optical modes as well as in SEM due to their topographic contrast (Fig. 4)
Summary
For three-dimensional (3D) ultrastructural investigations with electron microscopy (EM), five different techniques are typically used (Fig. 1) 1 μm); (iii) serial block face sectioning (3View®), a built-in ultramicrotome within a scanning electron microscopy (SEM), is suitable for large volumes but limited in section thickness (20 nm at best) and is hampered by charging; (iv) array tomography, which is non-destructive, but limited in z-resolution just as classical serial sectioning; and (v) focused ion beam (FIB)/SEMtomography, which currently offers by far the highest resolution along the z axis with a “section thickness” down to 2 nm for long image series (for review see: Peddie & Collinson, 2014; RomeroBrey & Bartenschlager, 2015; Karreman et al, 2016; Xu et al., 2017). The use of different detectors and the variation of many SEM parameters offer enormous analytical capacities, well beyond surface imaging
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