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

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.

Insights into replicative senescence of human testicular peritubular cells

“…Cells were seeded on laser marked slides 69 . For light microscopic investigations cells were fixed in cacodylate buffer and stained with DAPI, as described by Luckner and Wanner 69 .…”

“…Cells were seeded on laser marked slides 69 . For light microscopic investigations cells were fixed in cacodylate buffer and stained with DAPI, as described by Luckner and Wanner 69 . From each passage 10 representative interphase cells were selected in phase contrast light microscopy according to their cell size and shape and documented with a CCD camera with the corresponding epifluorescence DAPI image.…”

“…From each passage 10 representative interphase cells were selected in phase contrast light microscopy according to their cell size and shape and documented with a CCD camera with the corresponding epifluorescence DAPI image. Cells were post-fixed/stained with either osmium tetroxide or reduced osmium-TCH-osmium (=rOTO) and ultra-thin embedded with epoxy resin as described 36,69 . After polymerization, the cells were documented again by bright field light microscopy for visualization and control of the heavy metal staining again.…”

“…After polymerization, the cells were documented again by bright field light microscopy for visualization and control of the heavy metal staining again. For high resolution SEM and FIB milling, cells were processed as described in detail by Luckner and Wanner 69 . Images were recorded with 3072 × 2048 pixel.…”

Volume electron microscopy: analyzing the lung

“…FIB-SEM has the great advantage over SBF-SEM to be applicable on archive material conventionally embedded for TEM, as high-contrast achieving protocols like rOTO (see above) are not required (Hegermann et al 2019 ; Steyer et al 2020 ; Wrede et al 2020 ). Similar to SBF-SEM, however, the resin block surface is made conductive by coating it with metals and carbon (Luckner and Wanner 2018 ; Wrede et al 2020 ), but while in SBF-SEM this coat is removed by the diamond knife, in a FIB-SEM, the resin block stays coated and only the small FOV of a few hundred µm 2 is unsealed by the FIB. In this way, charging effects are minimized, making imaging much more feasible compared to SBF-SEM.…”