High-resolution Light Microscopy Research Articles

Cover Feature: The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System (ChemBioChem 18/2021)

High-resolution light microscopy of Helicobacter pylori-infected gastric tissue (400× magnification): The picture shows a composite fluorescent image of gastric tissue stained with eosin (pseudo-colored blue) producing lactoferrin (pseudocolored overlay cyan), with the structure of lactoferrin in an inset (circular panel). H. pylori is a Gram-negative bacterial pathogen that infects the stomach and causes ulcers and gastric cancer. The host innate immune system produces the glycoprotein lactoferrin as part of the antimicrobial response to bacterial infection, and this molecule can repress the oncogenic activity of the H. pylori cag T4SS in an iron-dependent manner. More information can be found in the Full Paper by S. D. Townsend, J. A. Gaddy et al.

Epiblast and trophoblast morphogenesis in the pre-gastrulation blastocyst of the pig. A light- and electron-microscopical study.

The epiblast of the amniote embryo is of paramount importance during early development as it gives rise to all tissues of the embryo proper. In mammals, it emerges through segregation of the hypoblast from the inner cell mass and subsequently undergoes transformation into an epithelial sheet to create the embryonic disc. In rodents and man, the epiblast cell layer is covered by the polar trophoblast which forms the placenta. In mammalian model organisms (rabbit, pig, several non-human primates), however, the placenta is formed by mural trophoblast whereas the polar trophoblast disintegrates prior to gastrulation and thus exposes the epiblast to the microenvironment of the uterine cavity. Both, polar trophoblast disintegration and epiblast epithelialization, thus pose special cell-biological requirements but these are still rather ill-understood when compared to those of gastrulation morphogenesis. This study therefore applied high-resolution light and transmission electron microscopy and three-dimensional (3D) reconstruction to 8- to 10-days-old pig embryos and defines the following steps of epiblast transformation: (1) rosette formation in the center of the ball-shaped epiblast, (2) extracellular cavity formation in the rosette center, (3) epiblast segregation into two subpopulations - addressed here as dorsal and ventral epiblast - separated by a "pro-amniotic" cavity. Ventral epiblast cells form between them a special type of desmosomes with a characteristic dense felt of microfilaments and are destined to generate the definitive epiblast. The dorsal epiblast remains a mass of non-polarized cells and closely associates with the disintegrating polar trophoblast, which shows morphological features of both apoptosis and autophagocytosis. Morphogenesis of the definitive epiblast in the pig may thus exclude a large portion of bona fide epiblast cells from contributing to the embryo proper and establishes contact de novo with the mural trophoblast at the junction between the two newly defined epiblast cell populations.

Effect of fullerite powder size on the properties of composite materials synthesized at high pressure from iron-C60/70 mixtures

Fullerite C60/70 particles of > 315, 315 – 125, 125–60, and < 60 μm in size were used for synthesis of composite materials (CM) from their mixtures with iron powder by heating to temperatures of 1200 – 1400 K at a pressure of 5 GPa. The effect of fullerite particle size in iron matrix on the formation of superelastic hard carbon phase after fullerene molecule collapse has been studied by the methods of high-resolution Raman spectroscopy, light microscopy, and microindentation. The coarsest particles prepared at 1200 K retain polymerized fullerites, while the finest particles prepared at 1400 K exhibit partial graphitization. Tribological tests showed that the wear resistance of the CM is determined by the degree of transformation completion, and the reinforcement effect increases with sand-paper grain refinement. Graphitization of the reinforcing particles leads to a decrease in wear resistance (by a factor of ∼3) at a retention of a relatively low friction coefficient. The most stable results (high wear resistance and friction coefficient of 0.18–0.3) are characteristic of the CM reinforced with carbon particles prepared from fullerites of medium size.

Differentiation of osteoblast lineage cells that form dermal bones and sutures of the anterior cranial vault in development and disease

FGFR-related craniosynostosis syndromes, such as Apert syndrome, are characterized in part by dysmorphology of the anterior cranial vault bones and early closure of the coronal suture. Understanding the cellular processes during early formation of the anterior cranial vault bones and the coronal suture is key to the development of therapies for these syndromes. Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) play critical roles in the regulation of osteoblast differentiation and function and are first expressed at different stages of osteoblast lineage cell (OLC) development. RUNX2 is known to act upstream of OSX and is expressed during an earlier stage of OLC differentiation, but relatively little is known about the behavior and developmental sequence of OLCs that contribute to the initial formation of cranial dermal bones and the sutures between them. To address this issue, we made the R2Tom transgenic mouse line by introducing the tdTomato reporter gene joined with an enhancer fragment of the human RUNX2 gene and the Hsp68 minimal promoter sequence that labels developmentally early OLCs. Crossing this novel R2Tom mouse line with the Osx-GFP line produced R2Tom;Osx-GFP mice, in which expression of RUNX2 and Osx is detected with different fluorescent reporters. The R2Tom;Osx-GFP mouse line was then crossed with Fgfr2+/P253R;Emx1-Cre (referred to below as Apert) mice that show phenotypes similar to Apert syndrome patients to produce Apert;R2Tom;Osx-GFP mice and “unaffected” littermates that carry the fluorescent marker but do not express the mutant Fgfr2 gene. Using large field, high-resolution fluorescent microscopy (ranging between 12.5x and 112x) and light sheet microscopy, we captured expression of RUNX2 and Osx across the surface of whole mouse embryos and at the cellular level. R2Tom;Osx-GFP and Apert;R2Tom;Osx-GFP mice were developmentally staged to provide precise information about the developmental sequence of expression of RUNX2 and Osx and 3D physical organization of early OLCs during the cellular assembly of what will be the frontal and parietal bones and initiation of the coronal suture in mouse embryos between E11.5 to E14.5. Our preliminary data on Apert;R2Tom;Osx-GFP mice at E12.5 and E13.5 reveal differences in the timing and pattern of the developmental sequence of RUNX2 and Osx expression in the anterior cranial vault of mice carrying the Fgfr2+/P253R mutation relative to their unaffected litter mates. These results provide our first view of OLC differentiation in the anterior cranial vault of Fgfr2+/P253R Apert mice prior to the formation of anterior cranial vault bones and the coronal suture and suggest that differences in the timing of OLC patterning play an import role in dysmorphologies of the anterior cranial vault.

Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington\u2019s disease

Huntington’s disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

Application of Advanced Light Microscopy to the Study of HIV and Its Interactions with the Host.

This review highlights the significant observations of human immunodeficiency virus (HIV) assembly, release and maturation made possible with advanced light microscopy techniques. The advances in technology which now enables these light microscopy measurements are discussed with special emphasis on live imaging approaches including Total Internal Reflection Fluorescence (TIRF), high-resolution light microscopy techniques including PALM and STORM and single molecule measurements, including Fluorescence Resonance Energy Transfer (FRET). The review concludes with a discussion on what new insights and understanding can be expected from these measurements.

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To date, some species in the genus Thyreophagus, such as Th. entomophagus and Th. cracentiseta, have been shown to be useful factitious prey for mass-rearing of predacious phytoseiid mites, one of the most common groups of mites used for biological control. Here, we describe a new species, Thyreophagus plocepasseri sp. n. from Kenya that can serve as a factitious prey as well. In addition, the new species is asexual, a trait that can be advantageous for its mass-rearing in industrial settings. In the description, we use high-resolution light microscope imaging and extensive comparison with a similar species, Thyreophagus athiasae, for which the type was studied. We review the literature on asexual acarid mites and point to a lack of data on the presence of sex-manipulating bacteria (Wolbachia, Cardinium) in these asexual mite lineages. Based on the literature, these bacteria are known to induce parthenogenesis in several haplodiploid mites, but they have not been linked so far to parthenogenesis inducement in diplodiploid acarid mites.

A workflow for 3D-CLEM investigating liver tissue.

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10-9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.

A correlation analysis of Light Microscopy and X-ray MicroCT imaging methods applied to archaeological plant remains\u2019 morphological attributes visualization

In this work, several attributes of the internal morphology of drupaceous fruits found in the archaeological site Monte Castelo (Rondonia, Brazil) are analyzed by means of two different imaging methods. The aim is to explore similarities and differences in the visualization and analytical properties of the images obtained via High Resolution Light Microscopy and X-ray micro-computed tomography (X-ray MicroCT) methods. Both provide data about the three-layered pericarp (exo-, meso- and endocarp) of the studied exemplars, defined by cell differentiation, vascularisation, cellular contents, presence of sclerenchyma cells and secretory cavities. However, it is possible to identify a series of differences between the information that can be obtained through each of the methods. These variations are related to the definition of contours and fine details of some characteristics, their spatial distribution, size attributes, optical properties and material preservation. The results obtained from both imaging methods are complementary, contributing to a more exhaustive morphological study of the plant remains. X-ray MicroCT in phase-contrast mode represents a suitable non-destructive analytic technique when sample preservation is required.

Reprint of: The effects of decomposition and environment on antemortem H-Pb-Sr isotope compositions and degradation of human scalp hair: Actualistic taphonomic observations

Multi-isotope analysis (e.g., Sr-Pb-O-H-C-N) of human scalp hair is routinely used in forensic investigations of human remains to constrain the geographic origin of unidentified bodies, and to investigate antemortem mobility patterns. However, while it is known that postmortem processes can affect the preservation of, or even overprint, the biogenic isotopic signatures in hair, the speed and nature of these processes have rarely been studied. This study investigates the effects of decomposition and environment on the H-Pb-Sr isotope compositions of human hair as well as the relationship between structural hair shaft degradation and isotopic signature change over time. Human scalp hair samples from four body donations were collected at different stages throughout gross body decomposition. The willed-donated bodies were placed to decompose outdoors at the Forensic Anthropology Research Facility (FARF) at Texas State University. Hair fibres from two of the donations were examined using scanning electron microscopy (SEM) and high-resolution light microscopy (HRLM). Chemical and microbiological degradation of hair fibres occurred rapidly after placement of the body outdoors. Measurements of scalp hair isotopic composition demonstrated that H-Pb-Sr isotope ratios were altered within days after environmental exposure, presumably by deposition, leaching and/or exchange with the local bioavailable soil, and vapour. The degree of physical hair degradation and changes in H-Pb-Sr isotope composition were not correlated. We conclude that antemortem isotopic H-Pb-Sr isotope ratios are difficult to recover in hairs derived from decomposing whole bodies.

Second Harmonic Generation Imaging of Collagen in Chronically Implantable Electrodes in Brain Tissue.

Advances in neural engineering have brought about a number of implantable devices for improved brain stimulation and recording. Unfortunately, many of these micro-implants have not been adopted due to issues of signal loss, deterioration, and host response to the device. While glial scar characterization is critical to better understand the mechanisms that affect device functionality or tissue viability, analysis is frequently hindered by immunohistochemical tissue processing methods that result in device shattering and tissue tearing artifacts. Devices are commonly removed prior to sectioning, which can itself disturb the quality of the study. In this methods implementation study, we use the label free, optical sectioning method of second harmonic generation (SHG) to examine brain slices of various implanted intracortical electrodes and demonstrate collagen fiber distribution not found in normal brain tissue. SHG can easily be used in conjunction with multiphoton microscopy to allow direct intrinsic visualization of collagen-containing glial scars on the surface of cortically implanted electrode probes without imposing the physical strain of tissue sectioning methods required for other high resolution light microscopy modalities. Identification and future measurements of these collagen fibers may be useful in predicting host immune response and device signal fidelity.

A case of morphological anomalies in Amblyomma americanum (Acari: Ixodidae) collected from nature

We describe a case of morphological anomalies in Amblyomma americanum, a medically important species associated with several human diseases and medical conditions. Based on morphological characters using dichotomous morphological keys, high-resolution light microscopy, and scanning electron microscopy imaging, the tick was identified as Am. americanum nymph exhibiting various morphological anomalies including ectromely associate with asymmetry, olygomely (lack) of the fourth left leg, and schizomely (bifurcation of palpus) on the right side. We believe this is the first report of the presence of several spontaneous anomalies in one Am. americanum specimen. Morphological identity of the specimen was corroborated by DNA sequencing of the mitochondrial 16S region. We discuss recent reports of morphological anomalies in ixodid ticks and emphasize the significance of additional studies of teratology in medically important tick species and its potential implications.

The effects of decomposition and environment on antemortem H-Pb-Sr isotope compositions and degradation of human scalp hair: Actualistic taphonomic observations

Multi-isotope analysis (e.g., Sr–Pb–O–H–C–N) of human scalp hair is routinely used in forensic investigations of human remains to constrain the geographic origin of unidentified bodies, and to investigate antemortem mobility patterns. However, while it is known that postmortem processes can affect the preservation of, or even overprint, the biogenic isotopic signatures in hair, the speed and nature of these processes have rarely been studied. This study investigates the effects of decomposition and environment on the H–Pb–Sr isotope compositions of human hair as well as the relationship between structural hair shaft degradation and isotopic signature change over time. Human scalp hair samples from four body donations were collected at different stages throughout gross body decomposition. The willed-donated bodies were placed to decompose outdoors at the Forensic Anthropology Research Facility (FARF) at Texas State University. Hair fibers from two of the donations were examined using scanning electron microscopy (SEM) and high-resolution light microscopy (HRLM). Chemical and microbiological degradation of hair fibers occurred rapidly after placement of the body outdoors. Measurements of scalp hair isotopic composition demonstrated that H–Pb–Sr isotope ratios were altered within days after environmental exposure, presumably by deposition, leaching and/or exchange with the local bioavailable soil, and vapor. The degree of physical hair degradation and changes in H–Pb–Sr isotope composition were not correlated. We conclude that antemortem isotopic H–Pb–Sr isotope ratios are difficult to recover in hairs derived from decomposing whole bodies.

Drug Permeability Profiling Using the Novel Permeapad® 96-Well Plate.

Here, first experiences with a prototype tool for high throughput (passive) permeability profiling, a 96-well plate comprising the Permeapad® membrane, are reported. The permeabilities of a set of drugs were determined and compared to published measures of oral absorption, such as human fraction absorbed (Fa) and in vitro permeability values obtained using other tools. The tool consists of a 96-well bottom and screen plate with the artificial, phospholipid-based barrier (Permeapad®) mounted between the plates' lower and upper compartments. The permeability of 14 model compounds including high- and low-absorption drugs, cationic, anionic, zwitterionic and neutral molecules, was determined by quantifying the compounds' transport over time, deriving the steady-state flux from the linear part of the cumulative curves and calculating the apparent permeability (Papp). The membrane structure was investigated in a high-resolution digital light microscope. The Permeapad® 96-well plate was found suited to distinguish high and low absorption drugs and yielded a hyperbolic correlation to Fa. The Papp values obtained were congruent with those determined with in-house prepared Permeapad® in the Franz cell set-up. Furthermore, good to excellent correlations were seen with Caco-2 permeability (R2= 0.70) and PAMPA permeability (R2= 0.89). Microscopic investigation of the Permeapad® barrier revealed the formation of phospholipid vesicles and myelin figures in aqueous environment. The Permeapad® 96-well plate permeation set-up is a promising new tool for rapid and reproducible passive permeability profiling.

Visualizing the genome in high resolution challenges our textbook understanding.

The relationship between the 4D folding of the genome and its function is an outstanding question in biology. A range of methods that probe the folding of the genome in space and time with unprecedented resolution have been developed. These methods, including chromosome conformation capture and high-resolution light and electron microscopy, are shedding new light on genome architecture and function. Here, we review the emerging picture of genome organization revealed by super-resolution and live-cell imaging. We compare and contrast population-based chromosome conformation capture approaches and imaging-based approaches and highlight future challenges.

Spatial relationship between telocytes, interstitial cells of Cajal and the enteric nervous system in the human ileum and colon.

Telocytes (TCs) are recently described interstitial cells, present in almost all human organs. Among many other functions, TCs regulate gastrointestinal motility together with the interstitial cells of Cajal (ICCs). TCs and ICCs have close localization in the human myenteric plexus; however, the exact spatial relationship cannot be clearly examined by previously applied double immunofluorescence/confocal microscopy. Data on TCs and submucosal ganglia and their relationship to intestinal nerves are scarce. The aim of the study was to analyse the spatial relationship among these components in the normal human ileum and colon with double CD34/CD117 and CD34/S100 immunohistochemistry and high‐resolution light microscopy. TCs were found to almost completely encompass both myenteric and submucosal ganglia in ileum and colon. An incomplete monolayer of ICCs was localized between the TCs and the longitudinal muscle cells in ileum, whereas only scattered ICCs were present on both surfaces of the colonic myenteric ganglia. TC‐telopodes were observed within colonic myenteric ganglia. TCs, but no ICCs, were present within and around the interganglionic nerve fascicles, submucosal nerves and mesenterial nerves, but were only observed along small nerves intramuscularly. These anatomic differences probably reflect the various roles of TCs and ICCs in the bowel function.

Chiral Os(II) Polypyridyl Complexes as Enantioselective Nuclear DNA Imaging Agents Especially Suitable for Correlative High-Resolution Light and Electron Microscopy Studies.

The high-resolution technique transmission electron microscopy (TEM), with OsO4 as the traditional fixative, is an essential tool for cell biology and medicine. Although OsO4 has been extensively used, it is far from perfect because of its high volatility and toxicity. Os(II) polypyridyl complexes like [Os(phen)2(dppz)]2+ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) are not only the well-known molecular DNA "light-switches" but also the potential ideal candidates for TEM studies. Here, we report that the cell-impermeable cationic [Os(phen)2(dppz)]2+ can be preferentially delivered into the live-cell nucleus through ion-pairing with chlorophenolate counter-anions, where it functions as an unparalleled enantioselective nuclear DNA imaging reagent especially suitable for correlative light and electron microscopy (CLEM) studies in both living and fixed cells, which can clearly visualize chromosome aggregation and decondensation during mitosis simultaneously. We propose that the chiral Os(II) polypyridyl complexes can be used as a distinctive group of enantioselective high-resolution CLEM imaging probes for live-cell nuclear DNA studies.

Saccharomyces cerevisiae RC016 modulates the apoptotic pathways in rat livers treated with aflatoxin B1

The aim was to study the effect of probiotic Saccharomyces cerevisiae RC016 on the expression of apoptotic protein Bax, Bcl-2, DR4 and c-FLIP, in liver of rats exposed to aflatoxin B1 (AFB1). Four treatments were applied to inbred male Wistar rats: uncontaminated feed control, S. cerevisiae RC016 control, contaminated feed with 100 μg/kg AFB1 and contaminated feed with 100 μg/kg AFB1 + daily oral dose 108 viable S. cerevisiae RC016 cells. Histological technique and high-resolution light microscopy (HRLM) were performed to the study of tissue morphology, the TUNEL assay was used to determine the apoptosis cellular and the expression of Bax, Bcl-2, DR4 and c-FLIP was determinate through immunohistochemistry. In liver the necrotic lesions observed with AFB1 treatment were reduced with the addition of yeast. The highest apoptotic index (IAp) was found in the yeast control, with AFB1 decrease significantly the IAp, while with the addition of yeast increase the IAp of liver cells. This was confirmed by HRLM. DR4 receptor was not present in any of the treatments. The immunolabeling of c-FLIP showed a statistically significant increase in the treatments with S. cerevisiae. The extrinsic pathway of apoptosis through the FAS-receptors would neither be active in the apoptotic process observed in rat livers in the treatments with yeast. Significant differences between proteins Bax and Bcl-2 and effect of treatments on the immunolabeling of Bax were determinate. The exposure to AFB1 decreased the IAp in the livers; while the addition of the yeast produced a significant statistically increase of IAp. In this study it was determined that the apoptosis in liver would be induced by the intrinsic pathway through Bax. These suggest that the incorporation of the autocrine strain S. cerevisiae RC016 increases the apoptosis in liver, counteracting the adverse effect of aflatoxin B1 and favouring the tissue remodulation.

Mechanical impact stimulation platform tailored for high-resolution light microscopy

High frequency (HF) mechanical vibration has been used in vitro to study the cellular response to mechanical stimulation and induce stem cell differentiation. However, detailed understanding of the effect of the mechanical cues on cellular physiology is lacking. To meet this limitation, we have designed a system, which enables monitoring of living cells by high-resolution light microscopy during mechanical stimulation by HF vibration or mechanical impacts. The system consists of a commercial speaker, and a 3D printed sample vehicle and frame. The speaker moves the sample in the horizontal plane, allowing simultaneous microscopy. The HF vibration (30–200 Hz) performances of two vehicles made of polymer and aluminum were characterized with accelerometer. The mechanical impacts were characterized by measuring the acceleration of the aluminum vehicle and by time lapse imaging. The lighter polymer vehicle produced higher HF vibration magnitudes at 30–50 Hz frequencies than the aluminum vehicle. However, the aluminum vehicle performed better at higher frequencies (60–70 Hz, 90–100 Hz, 150 Hz). Compatibility of the system in live cell experiments was investigated with epithelial cells (MDCKII, expressing Emerald-Occludin) and HF (0.56 Gpeak, 30 Hz and 60 Hz) vibration. Our findings indicated that our system is compatible with high-resolution live cell microscopy. Furthermore, the epithelial cells were remarkable stable under mechanical vibration stimulation. To conclude, we have designed an inexpensive tool for the studies of cellular biophysics, which combines versatile in vivo like mechanical stimuli with live cell imaging, showing a great potential for several cellular applications.

High-resolution computed tomography reveals dynamics of desiccation and rehydration in fern petioles of a desiccation-tolerant fern.

Desiccation-tolerant (DT) plants can dry past -100MPa and subsequently recover function upon rehydration. Vascular DT plants face the unique challenges of desiccating and rehydrating complex tissues without causing structural damage. However, these dynamics have not been studied in intact DT plants. We used high resolution micro-computed tomography (microCT), light microscopy, and fluorescence microscopy to characterize the dynamics of tissue desiccation and rehydration in petioles (stipes) of intact DT ferns. During desiccation, xylem conduits in stipes embolized before cellular dehydration of living tissues within the vascular cylinder. During resurrection, the chlorenchyma and phloem within the stipe vascular cylinder rehydrated before xylem refilling. We identified unique stipe traits that may facilitate desiccation and resurrection of the vascular system, including xylem conduits containing pectin (which may confer flexibility and wettability); chloroplasts within the vascular cylinder; and an endodermal layer impregnated with hydrophobic substances that impede apoplastic leakage while facilitating the upward flow of water within the vascular cylinder. Resurrection ferns are a novel system for studying extreme dehydration recovery and embolism repair in the petioles of intact plants. The unique anatomical traits identified here may contribute to the spatial and temporal dynamics of water movement observed during desiccation and resurrection.