Bruno M. Humbel

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy).Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ‐H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ‐H2AX a round, electron‐dense nuclear structure was found, which was hitherto not identified in UV‐stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy‐loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV‐induced nuclear structure contains a high amount of RNA.Conclusions. Because the UV‐induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well‐known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV‐induced apoptotic pathway.

Summary: Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three‐dimensional micro‐ and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block‐face imaging using low‐kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.

Epidermal growth factor (EGF) receptor RNA has been shown to be localized around nucleoli in the nucleus of A431 cells (Sibon et al., Histochemistry 101, 223-232 (1994)). Here we have studied the functional implication of this localization. Inhibition of transcription by alpha-amanitin did not influence the localization and amount of EGF-receptor RNA around the nucleolus, indicating that these RNAs represent mainly completed transcripts. Localization of the EGF-receptor genes in A431 cells by in situ hybridization revealed that the majority of the receptor gene clusters are located at the periphery of the nucleus. Next to this virtually all cells studied contain at least one gene cluster in the vicinity of the nucleolus. From these data, it is tempting to suggest that EGF-receptor gene transcription occurs around the nucleolus. In order to obtain information on the site of EGF-receptor RNA splicing, the localization of exon and intron sequences of the EGF-receptor transcripts was studied using a new electron microscopical approach. These labeling studies revealed that both intron and exon sequences were present at the same site around the nucleolus. In addition, exon sequences were also located, around nucleolus separate from intron sequences. All together, these studies suggest that transcription and splicing of the EGF-receptor transcript occurs at the same defined site around the nucleolus in A431 cells.