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

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.

DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei.

Replication protein A (RP-A) is involved in DNA replication, repair and recombination. It has been demonstrated that RP-A clusters in foci prior to DNA replication and redistributes over chromatin during S-phase. Here, we show that RP-A foci also form in response to DNA double-strand (ds) breaks produced on Xenopus laevis sperm nuclei by restriction enzymes and then reconstituted with Xenopus egg high-speed extracts. Ku86 co-localizes with RP-A in the same foci. An unscheduled RP-A-dependent DNA synthesis takes place overlapping with RP-A and Ku86 foci. Immunoelectron-microscopy analysis reveals that these foci correspond to spherical bodies up to 300 nm in diameter, which contain RP-A, Ku86 and DNA. In an independent in vitro assay, we incubated linear dsDNA bound to magnetic beads with Xenopus egg extracts. Here, also RP-A and Ku cluster in foci as seen through immunofluorescence. Both proteins appear to enrich themselves in sequences near the ends of the DNA molecules and influence ligation efficiency of ds linear DNA to these ends. Thus, the Xenopus in vitro system allows for the generation of specific DNA ds breaks, RP-A and Ku can be used as markers for these lesions and the repair of this type of DNA damage can be studied under conditions of a normal nuclear environment.

Ultrastructural characterization of RPA-containing domains in nuclei assembled in Xenopus egg extracts.

We describe novel structural domains in in vitro reconstituted Xenopus sperm nuclei, which we term RPA bodies; RPA is the only known marker of these structures. These bodies contain DNA and represent special chromatin domains as seen by transmission electron microscopy. We show that RPA bodies exhibit a similar ultrastructure in nuclei assembled in high-speed supernatant (HSS) of Xenopus egg extract and in nuclei assembled in HSS supplemented with low-speed supernatant (HSS + LSS nuclei). Moreover, RPA bodies are also formed when sperm chromatin containing double-stranded DNA breaks is incubated with HSS of egg extracts. RPA bodies appear to be compartmentalized. By immunoelectron microscopy we show that RPA is preferentially localized at the periphery of the bodies where DNA synthesis also occurs in HSS + LSS nuclei.

Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.

A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the increase in cell membrane permeability under hypotonic conditions is a general phenomenon in all animal cells tested. The efficiency of the method depends on the composition and temperature of the hypotonic buffer, the duration of the hypotonic treatment and the molecular weight of the molecules introduced into living cells. The versatility of this approach is demonstrated with various types of molecules such as modified nucleotides, nucleotides with conjugated fluorochrome, peptides, phosphatase substrates and fluorescent dyes. The method opens new possibilities for the direct investigation of a variety of biological problems as documented here with data on the functional organization of the cell nucleus.

Non-isotopic detection of nucleolar transcription in pre-implantation mouse embryos.

Nucleolar transcription was analysed in permeabilized pre-implantation mouse embryos at the four-cell, eight-cell, morula and early blastocyst stages using confocal microscopy to detect incorporated 5-bromouridine. The results demonstrated that the patterns of nucleolar transcription sites were common for all embryonic stages studied. They consisted most frequently of tightly associated groups of transcription foci similar to those encountered in somatic interphase cells. In addition, the nucleologenesis accompanying each cell cycle apparently gave rise to a different fluorescent pattern, that is to spatially separated fluorescent foci in the cells just after the resumption of rRNA synthesis. An immunoelectron microscopic analysis of the nucleolar transcription was also performed in the eight-cell embryos. A signal, usually consisting of clustered gold particles, was found specifically within nucleolar dense fibrillar components. This result was in agreement with established findings, which identify dense fibrillar component as the major site of nucleolar transcription in somatic cells.