From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References

Luckner, Manja und Wanner, Gerhard (2018): From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References. In: Microscopy and Microanalysis, Bd. 24, Nr. 5: S. 526-544

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Biologie > Department Biologie I
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• ISSN: 1431-9276
• Sprache: Englisch