From light microscopy to analytical scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM in Biology: Fixed coordinates, flat embedding, absolute references

27Citations
Citations of this article
54Readers
Mendeley users who have this article in their library.

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.

Cite

CITATION STYLE

APA

Luckner, M., & Wanner, G. (2018). From light microscopy to analytical scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM in Biology: Fixed coordinates, flat embedding, absolute references. Microscopy and Microanalysis, 24(5), 526–544. https://doi.org/10.1017/S1431927618015015

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free