FIB‐SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20

Abstract

Summary Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high‐pressure freezing and freeze‐substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze‐substitution with per‐mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time‐efficient FIB‐SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM‐TEM workflows to comprehensively analyse biological structures. Lay Description Focused ion beam scanning electron microscopy is becoming a widely used technique for the three‐dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy‐based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub‐cellular and sub‐organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl‐embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures.

High-precision targeting workflow for volume electron microscopy

“…Our work shows that sample preparation protocols compatible with fluorescence preservation are satisfactory for FIB-SEM milling and imaging. In agreement with a previous report (Porrati et al, 2019), we achieved good contrast in the presence of 0.1% UA and complete absence of osmium. The stability and milling properties of Lowicryl HM20 under the FIB were comparable to the commonly used epoxy resins.…”

“…Recent publications showed good results in FIB-SEM acquisition of samples that were high-pressure frozen, freeze substituted (FS) with low amounts of UA, and embedded in acrylic resins (Höhn et al, 2015;Porrati et al, 2019). Based on these observations, we have established an easy and robust workflow that allows targeted FIB-SEM imaging of fluorescently labeled structures in a large volume (∼3.14 mm 2 × 0.2/0.4 mm depth).…”

Targeted volume Correlative Light and Electron Microscopy of an environmental marine microorganism

“…As most microalgae display characteristic autofluorescent properties, we decided to utilize light microscopy to target specific cells of interest. We therefore performed a freeze substitution method for preserving fluorescence by using a low amount of heavy metals and embedding in Lowicryl HM20 (Kukulski et al, 2011; Nixon et al, 2009; Porrati et al, 2019; Ronchi et al, 2021). Confocal 3D imaging of the resulting block revealed that the autofluorescence pattern was preserved after sample preparation and could be detected in the block for the entire thickness of the HPF material (200 µm) (Fig 1A, Video S1).…”

Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

“…Recent publications showed good results in FIB-SEM acquisition of samples that were high pressure frozen, freeze substituted with low amount of UA and embedded in acrylic resins (Höhn et al, 2015; Porrati et al, 2019). Based on these observations and on our previously established sample preparation protocols for on-section CLEM of tissues (Hampoelz et al, 2016, 2019; Wong et al, 2020; Lee et al, 2020), we have established an easy and robust workflow that allows targeted FIB-SEM imaging of fluorescently labelled structures in a large volume (~7mm 2 x 200/400 μm depth).…”