HighlightsUranyl acetate/tungsten double stains are proposed for imaging lipid rich nanoparticle in TEM.Combined with methylcellulose embedment, the technique enhances membrane contrast.The technique works for liposomes, nanodiscs and bicelles.The double staining should improve quantification of lipid rich nanoparticles.

Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5–10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell–cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

Microsporidia are obligate intracellular parasites causing significant disease in humans and economically important animals. In parallel to their extreme genetic reduction, Microsporidia have evolved novel mechanisms for exploiting host metabolism. A number of microsporidians confer secretion of otherwise cytosolic proteins by coding for signal peptides that direct entry into the endoplasmic reticulum. The human pathogen Trachipleistophora hominis encodes for four hexokinases, three of which have signal peptides at the N-terminus. Here, we localized hexokinase 2 and hexokinase 3 through developmental stages of T. hominis using light and electron microscopy. Both proteins were concentrated in an extracellular coat previously termed the plaque matrix (PQM). The PQM (containing hexokinases) was morphologically dynamic, infiltrating the host cytoplasm predominantly during replicative stages. Throughout development the PQM interacted closely with endoplasmic reticulum that was demonstrated to be active in membrane protein biosynthesis and export. The impact of hexokinase on the host metabolism was probed using the fluorescent analog of glucose, 2-NBDG, which displayed spatially restricted increases in signal intensity at the parasite/vacuole surface, coincident with hexokinase/PQM distribution. Gross metabolic aberrations, measured using metabolic profiling with the Seahorse XF Analyzer, were not detectable in mixed stage cocultures. Overall, these results highlight a role for the extended cell coat of T. hominis in host-parasite interactions, within which secreted hexokinases may work as part of a metabolic machine to increase glycolytic capacity or ATP generation close to the parasite surface.

Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM.

Anthropogenic noise is now a prominent pollutant increasing in both terrestrial and marine environments. In the ocean, proliferating offshore windfarms, a key renewable energy source, are a prominent noise concern, as their pile driving construction is among the most intense anthropogenic sound sources. Yet, across taxa, there is little information of pile driving noise impacts on organismal fine-scale movement despite its key link to individual fitness. Here, we experimentally quantified the swimming behavior of an abundant squid species (Doryteuthis pealeii) of vital commercial and ecological importance in response to in situ pile driving activity on multiple temporal and spatial scales (thus exposed to differing received levels, or noise-doses). Pile driving induced energetically costly alarm-jetting behaviors in most (69%) individuals at received sound levels (in zero to peak) of 112-123 dB re 1 µm s-2, levels similar to those measured at the kilometer scale from some wind farm construction areas. No responses were found at a comparison site with lower received sound levels. Persistence of swimming pattern changes during noise-induced alarm responses, a key metric addressing energetic effects, lasted up to 14 s and were significantly shorter in duration than similar movement changes caused by natural conspecific interactions. Despite observing dramatic behavioral changes in response to initial pile driving noise, there was no evidence of gait changes over an experiment day. These results demonstrate that pile driving disrupts squid fine-scale movements, but impacts are short-lived suggesting that offshore windfarm construction may minimally impact the energetics of this ecologically key taxon. However, further work is needed to assess potential behavioral and physiological impacts at higher noise levels.