Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.

Background Body size is a fundamental organismal trait. However, as body size and ecological contexts change across developmental time, evolutionary divergence may cause unexpected patterns of body size diversity among developmental stages. This may be particularly evident in polyphenic developmental stages specialized for dispersal. The dauer larva is such a stage in nematodes, and Caenorhabditis species disperse by traveling on invertebrate carriers. Here, we describe the morphology of a stress-resistant, dauer-like larval stage of the nematode Caenorhabditis inopinata, whose adults can grow to be nearly twice as long as its close relative, the model organism C. elegans. Results We find that a dauer-like, stress-resistant larval stage in two isolates of C. inopinata is on average 13% shorter and 30% wider than the dauer larvae of C. elegans, despite its much longer adult stage. Additionally, many C. inopinata dauer-like larvae were ensheathed, a possible novelty in this lineage reminiscent of the infective juveniles of parasitic nematodes. Variation in dauer-like larva formation frequency among twenty-four wild isolates of C. inopinata was also observed, although frequencies were low across all isolates (< 2%), with many isolates unable to produce dauer-like larvae under conventional laboratory conditions. Conclusion Most Caenorhabditis species thrive on rotting plants and disperse on snails, slugs, or isopods (among others) whereas C. inopinata is ecologically divergent and thrives in fresh Ficus septica figs and disperses on their pollinating wasps. While there is some unknown factor of the fig environment that promotes elongated body size in C. inopinata adults, the small size or unique life history of its fig wasp carrier may be driving the divergent morphology of its stress-resistant larval stages. Further characterization of the behavior, development, and morphology of this stage will refine connections to homologous developmental stages in other species and determine whether ecological divergence across multiple developmental stages can promote unexpected and opposing changes in body size dimensions within a single species.

BackgroundBody size is a fundamental organismal trait. However, as body size and ecological contexts change across developmental time, evolutionary divergence may cause unexpected patterns of body size diversity among developmental stages. This may be particularly evident in polyphenic developmental stages specialized for dispersal. The dauer larva is such a stage in nematodes, and Caenorhabditis species disperse by traveling on invertebrate carriers. Here, we describe the morphology of the dispersal dauer larva of the nematode Caenorhabditis inopinata, whose adults can grow to be nearly twice as long as its close relative, the model organism C. elegans.ResultsWe find that the C. inopinata dauer larva is shorter and fatter than those of its close relatives C. elegans, C. briggsae, and C. tropicalis, despite its much longer adult stage. Additionally, many C. inopinata dauer larvae were ensheathed, an apparent novelty in this lineage reminiscent of the infective juveniles of parasitic nematodes. We also found abundant variation in dauer formation frequency among twenty-four wild isolates of C. inopinata, with many strains unable to produce dauer larvae under laboratory conditions.ConclusionMost Caenorhabditis species thrive on rotting plants and disperse on snails, slugs, or isopods (among others) whereas C. inopinata is ecologically divergent and thrives in fresh Ficus septica figs and disperses on their pollinating wasps. These wasps are at least an order of magnitude smaller in length than the vectors of other Caenorhabditis species. While there is some unknown factor of the fig environment that promotes elongated body size in C. inopinata adults, the smaller size of its fig wasp carrier may be driving the reduced body length of its dauer larva. Thus ecological divergence across multiple developmental stages can promote unexpected and opposing changes in body size within a single species.

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