Determining the concentration of elements in subcellular structures poses a significant challenge. By locating an elemental species at high spatial resolution and with subcellular context, and subsequently quantifying it on an absolute scale, new information about cellular function can be revealed. Such measurements have not as yet been realised with existing techniques due to limitations on spatial resolution and inherent difficulties in detecting elements present in low concentrations. In this paper, we use scanning transmission electron microscopy (STEM) to establish a methodology for localising and quantifying high‐Z elements in a biological setting by measuring elastic electron scattering. We demonstrate platinum (Pt) deposition within neuronal cell bodies following in vivo administration of the Pt‐based chemotherapeutic oxaliplatin to validate this novel methodology. For the first time, individual Pt atoms and nanoscale Pt clusters are shown within subcellular structures. Quantitative measurements of elastic electron scattering are used to determine absolute numbers of Pt atoms in each cluster. Cluster density is calculated on an atoms‐per‐cubic‐nanometre scale, and used to show clusters form with densities below that of metallic Pt. By considering STEM partial scattering cross‐sections, we determine that this new approach to subcellular elemental detection may be applicable to elements as light as sodium.LAY DESCRIPTION: Heterogeneous elemental distributions drive fundamental biological processes within cells. While carbon, hydrogen, oxygen and nitrogen comprise by far the majority of living matter, concentrations and locations of more than a dozen other species must also be tightly controlled to ensure normal cell function. Oxaliplatin is a first‐line and adjuvant treatment for colorectal cancer. However, pain in the body's extremities (fingers and toes) significantly impairs clinical usage as this serious and persistent side effect impacts on both patient cancer care and quality of life. Annular dark‐field (ADF) imaging in the scanning transmission electron microscope (STEM) provides an image with strong atom‐number contrast and is sufficient to distinguish between different cell types and different organelles within the cells of the DRG. We also show that Pt may be imaged at the single atom level and be localised at very high resolution while still preserving a degree of ultrastructural context. The intrinsic image contrast generated is sufficient to identify these features without the need for heavy metal stains and other extensive processing steps which risk disturbing native platinum distributions within the tissue. We subsequently demonstrate that by considering the total elastic scattering intensity generated by nanometre‐sized Pt aggregations within the cell, the ADF STEM may be used to make a measurement of local concentration of Pt in units of atoms per cubic nanometre. We further estimate the minimum atomic number required to visualise single atoms in this setting, concluding that in similar samples it may be possible to detect species as light as sodium with atomic sensitivity.

High‐pressure freezing (HPF) is an electron microscopy (EM) preparation technique with superb ultrastructural preservation. Combined with EM tomography it provides virtual EM serial sections with extraordinary spatial resolution. For HPF, cells are usually cultured on a rigid sapphire disc that provides a tight fit in the holding bracket of the HPF apparatus. Since we are using extensible elastic silicone membranes as a growth support to perform cell stretch experiments, we developed a method to clamp the stretched silicone membrane and place it instead of the sapphire disc into the HPF holding bracket. Compared to chemical fixation the HPF immobilised cells showed improved structural preservation, partly even on a molecular level. However, the outstanding quality of HPF immobilised cells on sapphire discs was not achieved. Moreover, regions with obvious freezing artefacts seemed to be more abundant in the HPF silicone membranes, probably caused by lower heat transfer rates of the silicone membrane during the HPF process.Taken together, we have shown that HPF immobilisation can be performed on growth supports different than sapphire discs. Since even stretched membranes can be used with the new method, also other unconventional growth supports should not pose a problem.

Microscope objectives achieve near diffraction-limited performance only when used under the conditions they are designed for. In nonstandard geometries, such as thick cover slips or curved surfaces, severe aberrations arise, inevitably impairing high-resolution imaging. Correcting such large aberrations using standard adaptive optics can be challenging: existing solutions are either not suited for strong aberrations, or require extensive feedback measurements, consequently taking a significant portion of the photon budget. We demonstrate that it is possible to precompute the corrections needed for high-resolution imaging inside a glass tube based on a priori information only. Our ray-tracing-based method achieved over an order of magnitude increase in image contrast without the need for a feedbacksignal.

The past 20 years have seen a paradigm-shifting explosion of new optical microscopy technologies aimed at uncovering fundamental biological insights. Yet only a small portion 'cross the finish line' into wide adoption by the life science community. We contend that this is not primarily due to a lack of technical prowess or utility. Rather, many risks can conspire to derail the adoption of potentially disruptive technologies. One way to address these challenges is to de-risk paradigm-shifting inventions within open-access technology incubators. Here we detail the framework needed to shepherd innovative microscopy techniques through the often-treacherous adoption landscape to enable transformative scientific output.

Transmission electron microscopy can be used for the characterisation of a wide range of thin specimens, but soft matter and aqueous samples such as gels, nanoparticle dispersions, and emulsions will dry out and collapse under the microscope vacuum, therefore losing information on their native state and ultimately limiting the understanding of the sample. This study examines commonly used techniques in transmission electron microscopy when applied to the characterisation of cryogenically frozen Pickering emulsion samples. Oil-in-water Pickering emulsions stabilised by 3 to 5nm platinum nanoparticles were cryogenically frozen by plunge-freezing into liquid ethane to retain the native structure of the system without inducing crystallisation of the droplet oil cores. A comparison between the droplet morphology following different sample preparation methods has confirmed the effectiveness of using plunge-freezing to prepare these samples. Scanning transmission electron microscopy imaging showed that dry droplets collapse under the microscope vacuum, changing their shape and size (average apparent diameter: ∼342nm) compared to frozen samples (average diameter: ∼183nm). Cryogenic electron tomography was used to collect additional information of the 3D shape and size of the emulsion droplets, and the position of the stabilising nanoparticles relative to the droplet surface. Cryogenic energy dispersive X-ray and electron energy loss spectroscopy were used to successfully obtain elemental data and generate elemental maps to identify the stabilising nanoparticles and the oil phase. Elemental maps generated from spectral data were used in conjunction with electron tomography to obtain 3D information of the oil phase in the emulsion droplets. Beam-induced damage to the ice was the largest limiting factor to the sample characterisation, limiting the effective imaging resolution and signal-to-noise ratio, though careful consideration of the imaging parameters used allowed for the characterisation of the samples presented in this study. Ultimately this study shows that cryo-methods are effective for the representative characterisation of Pickering emulsions.

Ribosomes, discovered in 1955 by George Palade, were initially described as small cytoplasmic particles preferentially associated with the endoplasmic reticulum (ER). Over the years, extensive research has focused on both the structure and function of ribosomes. However, a fundamental question – how many ribosomes are present within whole cells – has remained largely unaddressed. In this study, we developed a microscopic method to quantify the total number of ribosomes in hTERT‐RPE‐1 cells and in nematode cells from various tissues of Caenorhabditis elegans hermaphrodites. Using electron tomography of high‐pressure frozen, freeze‐substituted and resin‐embedded samples, we determined that the ribosome number in hTERT‐RPE‐1 cells is in the same order of magnitude as biochemical measurements obtained via RNA capillary electrophoresis. As expected, control worms exhibited a higher number of ribosomes compared to RNA polymerase I A subunit (RPOA‐1)‐depleted worms in two out of three analysed tissue types. Our imaging‐based approach complements established biochemical methods by enabling direct quantification of ribosome numbers in specific samples. This method offers a powerful tool for advancing our understanding of ribosome localisation and distribution in cells and tissues across diverse model systems.

In this short and popular review, we summarise some of our findings analysing the replication cycles of large DNA viruses using scanning transmission electron tomography (STEM tomography) that we applied in the laboratory of Paul Walther. It is also a tribute to a very kind and expert scientist, who recently retired. Transmission electron microscopy (TEM), in particular cryo‐EM, has benefited tremendously from recent developments in instrumentation. However, TEM imaging remains limited by the thickness of the specimen and classical thin‐section TEM typically generates 2D representations of 3D volumes. Although TEM tomography can partly overcome this limitation, the thickness of the sample, the volume that can be analysed in 3D, remains limiting. STEM tomography can partly overcome this problem, as it allows for the analysis of thicker samples, up to 1 µm in thickness. As such, it is an interesting imaging technique to analyse large DNA viruses, some of which measure 1 µm or more, and which is the focus of our research interest.

It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application.The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit.The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques.Interdisciplinary research is welcome.Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens.