Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana.

Algal biomass is a viable source of chemicals and metabolites for various energy, nutritional, medicinal and agricultural uses. While stresses have commonly been used to induce metabolite accumulation in microalgae in attempts to enhance high-value product yields, this is often very detrimental to growth. Therefore, understanding how to modify metabolism without deleterious consequences is highly beneficial. We demonstrate that low-doses (1-5 Gy) of ionizing radiation in the X-ray range induces a non-toxic, hormetic response in microalgae to promote metabolic activation. We identify specific radiation exposure parameters that give reproducible metabolic responses in Chlorella sorokiniana caused by transcriptional changes. This includes up-regulation of >30 lipid metabolism genes, such as genes encoding an acetyl-CoA carboxylase subunit, phosphatidic acid phosphatase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase. The outcome is an increased lipid yield in stationary phase cultures by 25% in just 24 hours, without any negative effects on cell viability or biomass.

Universal pictures: A lithophane codex helps teenagers with blindness visualize nanoscopic systems.

People with blindness have limited access to the high-resolution graphical data and imagery of science. Here, a lithophane codex is reported. Its pages display tactile and optical readouts for universal visualization of data by persons with or without eyesight. Prototype codices illustrated microscopy of butterfly chitin-from N-acetylglucosamine monomer to fibril, scale, and whole insect-and were given to high schoolers from the Texas School for the Blind and Visually Impaired. Lithophane graphics of Fischer-Spier esterification reactions and electron micrographs of biological cells were also 3D-printed, along with x-ray structures of proteins (as millimeter-scale 3D models). Students with blindness could visualize (describe, recall, distinguish) these systems-for the first time-at the same resolution as sighted peers (average accuracy = 88%). Tactile visualization occurred alongside laboratory training, synthesis, and mentoring by chemists with blindness, resulting in increased student interest and sense of belonging in science.

Enhanced Resistance to Viruses in Nicotiana edwardsonii 'Columbia' Is Dependent on Salicylic Acid, Correlates with High Glutathione Levels, and Extends to Plant-Pathogenic Bacteria and Abiotic Stress.

Our earlier research showed that an interspecific tobacco hybrid (Nicotiana edwardsonii 'Columbia' [NEC]) displays elevated levels of salicylic acid (SA) and enhanced resistance to localized necrotic symptoms (hypersensitive response [HR]) caused by tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV), as compared with another interspecific hybrid (Nicotiana edwardsonii [NE]) derived from the same parents. In the present study, we investigated whether symptomatic resistance in NEC is indeed associated with the inhibition of TMV and TNV and whether SA plays a role in this process. We demonstrated that enhanced viral resistance in NEC is manifested as both milder local necrotic (HR) symptoms and reduced levels of TMV and TNV. The presence of an adequate amount of SA contributes to the enhanced defense response of NEC to TMV and TNV, as the absence of SA resulted in seriously impaired viral resistance. Elevated levels of subcellular tripeptide glutathione (GSH) in NEC plants in response to viral infection suggest that in addition to SA, GSH may also contribute to the elevated viral resistance of NEC. Furthermore, we found that NEC displays an enhanced resistance not only to viral pathogens but also to bacterial infections and abiotic oxidative stress induced by paraquat treatments. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Thylakoid Rhodanese-like Protein-Ferredoxin:NADP+ Oxidoreductase Interaction Is Integrated into Plant Redox Homeostasis System.

In vascular plants, the final photosynthetic electron transfer from ferredoxin (Fd) to NADP+ is catalyzed by the flavoenzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via an integral membrane protein TROL (thylakoid rhodanese-like protein) and the membrane associated protein Tic62. We have previously demonstrated that the absence of TROL triggers a very efficient superoxide (O2•-) removal mechanism. The dynamic TROL-FNR interaction has been shown to be an apparently overlooked mechanism that maintains linear electron flow before alternative pathway(s) is(are) activated. In this work, we aimed to further test our hypothesis that the FNR-TROL pair could be the source element that triggers various downstream networks of chloroplast ROS scavenging. Tandem affinity purification followed by the MS analysis confirmed the TROL-FNR interaction and revealed possible interaction of TROL with the thylakoid form of the enzyme ascorbate peroxidase (tAPX), which catalyzes the H2O2-dependent oxidation of ascorbate and is, therefore, the crucial component of the redox homeostasis system in plants. Further, EPR analyses using superoxide spin trap DMPO showed that, in comparison with the wild type, plants overexpressing TROL (TROL OX) propagate more O2•- when exposed to high light stress. This indicates an increased sensitivity to oxidative stress in conditions when there is an excess of membrane-bound FNR and less free FNR is found in the stroma. Finally, immunohistochemical analyses of glutathione in different Arabidopsis leaf cell compartments showed highly elevated glutathione levels in TROL OX, indicating an increased demand for this ROS scavenger in these plants, likely needed to prevent the damage of important cellular components caused by reactive oxygen species.

Plantorganelle Hunter is an effective deep-learning-based method for plant organelle phenotyping in electron microscopy.

Accurate delineation of plant cell organelles from electron microscope images is essential for understanding subcellular behaviour and function. Here we develop a deep-learning pipeline, called the organelle segmentation network (OrgSegNet), for pixel-wise segmentation to identify chloroplasts, mitochondria, nuclei and vacuoles. OrgSegNet was evaluated on a large manually annotated dataset collected from 19 plant species and achieved state-of-the-art segmentation performance. We defined three digital traits (shape complexity, electron density and cross-sectional area) to track the quantitative features of individual organelles in 2D images and released an open-source web tool called Plantorganelle Hunter for quantitatively profiling subcellular morphology. In addition, the automatic segmentation method was successfully applied to a serial-sectioning scanning microscope technique to create a 3D cell model that offers unique views of the morphology and distribution of these organelles. The functionalities of Plantorganelle Hunter can be easily operated, which will increase efficiency and productivity for the plant science community, and enhance understanding of subcellular biology.

A preparation of bacterial outer membrane with osmium tetroxide and uranyl acetate co-stain enables improved structural determination by transmission electron microscopy.

Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we report a novel method to prepare OMVs for TEM imaging. To preserve vesicular shape and structure, we developed a dual fixation protocol involving osmium tetroxide incubation prior to negative staining with uranyl acetate. Combining osmium tetroxide with uranyl acetate resulted in preservation of sub-50 nm vesicles and improved morphological stability, enhancing characterization of lipid-based nanoparticles by TEM.

A three-step process of manganese acquisition and storage in the microalga Chlorella sorokiniana.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese-calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Data for all: Tactile graphics that light up with picture-perfect resolution.

People who are blind do not have access to graphical data and imagery produced by science. This exclusion complicates learning and data sharing between sighted and blind persons. Because blind people use tactile senses to visualize data (and sighted people use eyesight), a single data format that can be easily visualized by both is needed. Here, we report that graphical data can be three-dimensionally printed into tactile graphics that glow with video-like resolution via the lithophane effect. Lithophane forms of gel electropherograms, micrographs, electronic and mass spectra, and textbook illustrations could be interpreted by touch or eyesight at ≥79% accuracy (n = 360). The lithophane data format enables universal visualization of data by people regardless of their level of eyesight.

Chemical and cryo-collection of muscle samples for transmission electron microscopy using Methacarn and dimethyl sulfoxide✰.

Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde and osmium tetroxide are widely used for transmission electron microscopy (TEM) and lead to adequate preservation of muscle ultrastructure, but do not preserve the molecular features of samples. Methacarn is suggested to be a preferable chemical fixative for light microscopy because it maintains immunohistological features of samples. However, the efficacy of methacarn to preserve ultrastructural features as a primary chemical fixative for TEM is currently unclear. Additionally, cryo-preservation of samples for TEM analysis involves freezing processes such as plunge freezing, slam freezing, or high pressure freezing. High pressure freezing is the considered the gold standard but requires costly equipment and may not be a viable option for many labs collecting tissue samples from remote locations. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant that may allow for better structural preservation of samples by impairing ice damage that occurs during plunge/snap freezing. We aimed to assess the effectiveness of methacarn as a primary chemical fixative and determine the effect of pre-coating samples with DMSO before plunge/snap freezing tissues to be prepared for TEM. The micrographs of the methcarn-fixed samples indicate a loss of Z-disk integrity, intermyofibrillar space, mitochondria structure, and lipids. Ultimately, methacarn is not a viable primary fixative for tissue sample preparation for TEM. Similarly, liquid nitrogen freezing of samples wrapped in aluminum foil produced non-uniform Z-disk alignments that appeared smeared with swollen mitochondria. DMSO coating before freezing appears to lessen the alterations to contractile and mitochondrial morphological structures. DMSO appears to be useful for preserving the ultrastructure of sarcomeres if samples are covered before freezing.

Conserved secreted effectors contribute to endophytic growth and multihost plant compatibility in a vascular wilt fungus.

Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.

Volumetric 3D reconstruction of plant leaf cells using SEM, ion milling, TEM, and serial sectioning.

MAIN CONCLUSION: Focused ion beam scanning electron microscopy is well suited for volumetric extractions and 3D reconstructions of plant cells and its organelles. The three-dimensional (3D) reconstruction of individual plant cells is an important tool to extract volumetric data of organelles and is necessary to fully understand ultrastructural changes and adaptations of plants to their environment. Methods such as the 3D reconstruction of cells based on light microscopical images often lack the resolution necessary to clearly reconstruct all cell compartments within a cell. The 3D reconstruction of cells through serial sectioning transmission electron microscopy (ssTEM) and focused ion beam scanning electron microscopy (FIB-SEM) are powerful alternatives but not widely used in plant sciences. Here, we present a method for the 3D reconstruction and volumetric extraction of plant cells based on FIB milling and compare the results with 3D reconstructions obtained with ssTEM. When compared to 3D reconstruction based on ssTEM, FIB-SEM delivered similar results. The data extracted in this study demonstrated that tobacco cells were larger (31410 µm3) than pumpkin cells (20697 µm3) and contained more chloroplasts (175 vs. 124), mitochondria (1317 vs. 291) and peroxisomes (745 vs. 79). While individual chloroplasts, mitochondria, peroxisomes were larger in pumpkin plants (25, 53, and 50%, respectively) they covered more total volume in tobacco plants (5390, 395, 374 µm3, respectively) due to their higher number per cell when compared to pumpkin plants (4762, 134, 59 µm3, respectively). While image acquisition with FIB-SEM was automated, software controlled, and less difficult than ssTEM, FIB milling was slower and sections could not be revised or re-imaged as they were destroyed by the ion beam. Nevertheless, the results in this study demonstrated that both, FIB-SEM and ssTEM, are powerful tools for the 3D reconstruction of and volumetric extraction from plant cells and that there were large differences in size, number, and organelle composition between pumpkin and tobacco cells.

Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum.

Root-infecting vascular fungi cause wilt diseases and provoke devastating losses in hundreds of crops. It is currently unknown how these pathogens evolved and whether they can also infect nonvascular plants, which diverged from vascular plants over 450 million years ago. We established a pathosystem between the nonvascular plant Marchantia polymorpha (Mp) and the root-infecting vascular wilt fungus Fusarium oxysporum (Fo). On angiosperms, Fo exhibits exquisite adaptation to the plant xylem niche as well as host-specific pathogenicity, both of which are conferred by effectors encoded on lineage-specific chromosomes. Fo isolates displaying contrasting lifestyles on angiosperms - pathogenic vs endophytic - are able to infect Mp and cause tissue maceration and host cell killing. Using isogenic fungal mutants we define a set of conserved fungal pathogenicity factors, including mitogen activated protein kinases, transcriptional regulators and cell wall remodelling enzymes, that are required for infection of both vascular and nonvascular plants. Markedly, two host-specific effectors and a morphogenetic regulator, which contribute to vascular colonisation and virulence on tomato plants are dispensable on Mp. Collectively, these findings suggest that vascular wilt fungi employ conserved infection strategies on nonvascular and vascular plant lineages but also have specific mechanisms to access the vascular niche of angiosperms.

Quantifying the effect of shade on cuticle morphology and carbon isotopes of sycamores: present and past.

PREMISE: Reconstructing the light environment and architecture of the plant canopy from the fossil record requires the use of proxies, such as those derived from cell wall undulation, cell size, and carbon isotopes. All approaches assume that plant taxa will respond predictably to changes in light environments. However, most species-level studies looking at cell wall undulation only consider "sun" or "shade" leaves; therefore, we need a fully quantitative taxon-specific method. METHODS: We quantified the response of cell wall undulation, cell size, and carbon isotopes of Platanus occidentalis using two experimental setups: (1) two growth chambers at low and high light and (2) a series of outdoor growth experiments using green and black shade cloth at different densities. We then developed and applied a proxy for daily light integral (DLI) to fossil Platanites leaves from two early Paleocene floras from the San Juan Basin in New Mexico. RESULTS: All traits responded to light environment. Cell wall undulation was the most useful trait for reconstructing DLI in the geological record. Median reconstructed DLI from early Paleocene leaves was ~44 mol m-2 d-1 , with values from 28 to 54 mol m-2 d-1 . CONCLUSIONS: Cell wall undulation of P. occidentalis is a robust, quantifiable measurement of light environment that can be used to reconstruct the paleo-light environment from fossil leaves. The distribution of high DLI values from fossil leaves may provide information on canopy architecture; indicating that either (1) most of the canopy mass is within the upper portion of the crown or (2) leaves exposed to more sunlight are preferentially preserved.

Cerebral Organoids Derived from a Parkinson's Patient Exhibit Unique Pathogenesis from Chikungunya Virus Infection When Compared to a Non-Parkinson's Patient.

(1) Background: Arboviruses of medical and veterinary significance have been identified on all seven continents, with every human and animal population at risk for exposure. Like arboviruses, chronic neurodegenerative diseases, like Alzheimer's and Parkinson's disease, are found wherever there are humans. Significant differences in baseline gene and protein expression have been determined between human-induced pluripotent stem cell lines derived from non-Parkinson's disease individuals and from individuals with Parkinson's disease. It was hypothesized that these inherent differences could impact cerebral organoid responses to viral infection. (2) Methods: In this study, cerebral organoids from a non-Parkinson's and Parkinson's patient were infected with Chikungunya virus and observed for two weeks. (3) Results: Parkinson's organoids lost mass and exhibited a differential antiviral response different from non-Parkinson's organoids. Neurotransmission data from both infected non-Parkinson's and Parkinson's organoids had dysregulation of IL-1, IL-10, and IL-6. These cytokines are associated with mood and could be contributing to persistent depression seen in patients following CHIKV infection. Both organoid types had increased expression of CXCL10, which is linked to demyelination. (4) Conclusions: The differential antiviral response of Parkinson's organoids compared with non-Parkinson's organoids highlights the need for more research in neurotropic infections in a neurologically compromised host.

Comparison of skeletal muscle ultrastructural changes between normal and blood flow-restricted resistance exercise: A case report.

NEW FINDINGS: What is the main observation in this case? The main observation of this case report is that blood flow-restricted exercise can cause myofibrils to have an aberrant wave-like appearance that is accompanied by irregular pockets of sarcoplasm in the intermyofibrillar space, while traditional forms of damage to the Z-discs and contractile elements are not as apparent. What insights does it reveal? Our findings indicate that blood flow restriction-mediated fluid pooling might cause alterations in skeletal muscle ultrastructure after exercise that might be directly related to myofibre swelling. ABSTRACT: The acute effects of blood flow-restricted (BFR) exercise training on skeletal muscle ultrastructure are poorly understood owing to inconsistent findings and the use of largely imprecise systemic markers for indications of muscle damage. The purpose of this study was to compare myofibrillar ultrastructure before and 30 min after normal and BFR resistance exercise using transmission electron microscopy in a single individual to evaluate the feasibility of this more nuanced approach. One apparently healthy male with 13 years of resistance exercise completed six sets of both BFR [30% of one-repetition maximum (1-RM)] and normal non-occluded (70% of 1-RM) unilateral angled leg press on the contralateral leg, as a control, after assessment of 1-RM 72 h before. Vastus lateralis muscle biopsies were collected before and 30 min after each exercise session. The lengths and widths of 250 sarcomeres and the sarcoplasmic area were assessed via 20 individual transmission electron photomicrographs. Analysis revealed that BFR training (1.769 ± 0.12 µm) increased sarcomere length when compared with normal exercise (1.64 ± 0.17 µm; P < 0.001), without differences in sarcomere width between conditions (BFR, 0.90 ± 0.26 µm; normal, 0.93 ± 0.27 µm; P = 0.172). Furthermore, there were no significant interaction (P = 0.168) or condition effects between BFR (25.98 ± 4.17%) and normal (27.3 ± 6.49%) resistance exercise for sarcoplasmic area (P = 0.229). Exercise also increased sarcoplasmic area within the myofibril (pre-exercise, 24.42 ± 5.13%; postexercise, 28.95 ± 5.92%) for both conditions (P = 0.001). This case study demonstrates a unique BFR training-induced alteration in myofibril ultrastructure that appeared wave like and was accompanied by intracellular abnormalities that appeared to be fluid pockets of sarcoplasm disrupting the surrounding myofibrils.

Three-dimensional quantitative imaging of Tobacco mosaic virus and Zucchini yellow mosaic virus induced ultrastructural changes.

Two-dimensional ultrastructural changes of Tobacco mosaic virus (TMV) and Zucchini yellow mosaic virus (ZYMV) in tobacco and pumpkin plants, respectively, are well studied. To provide 3D data, representative control and infected cells were reconstructed using serial sectioning and transmission electron microscopy. Quantitative data of 3D ultrastructural changes were then extracted from the cytosol and organelles by image analysis. While TMV induced the accumulation of an average of 40 virus inclusion bodies in the cytosol, which covered about 13% of the cell volume, ZYMV caused the accumulation of an average of 1752 cylindrical inclusions in the cytosol, which covered about 2.7% of the total volume of the cell. TMV infection significantly decreased the number and size of mitochondria (- 49 and - 20%) and peroxisomes (- 62 and - 28%) of the reconstructed cell. The reconstructed ZYMV-infected cell contained more (105%) and larger (109%) mitochondria when compared to the control cell. While the reconstructed TMV-infected cell contained larger (20%) and the ZYMV-infected smaller (19%) chloroplasts, both contained less chloroplasts (- 40% for TMV and - 23% for ZYMV). In chloroplasts, the volume of starch and plastoglobules increased (664% and 150% for TMV and 1324% and 1300% for ZYMV) when compared to the control. The latter was correlated with a decrease in the volume of thylakoids in the reconstructed ZYMV-infected cell (- 31%) indicating that degradation products from thylakoids are transported and stored in plastoglobules. Summing up, the data collected in this study give a comprehensive overview of 3D changes induced by TMV and ZYMV in plants.

Indoor Finish Material Influence on Contamination, Transmission, and Eradication of Methicillin-Resistant Staphylococcus aureus (MRSA).

OBJECTIVE: The purpose of this study was to evaluate environmental surface materials used in healthcare environments for material composition, methicillin-resistant Staphylococcus aureus (MRSA) viability, and a comparison of two disinfectants, a bleach germicidal cleaner and Decon7, a novel disinfectant. BACKGROUND: Contaminated environmental surfaces have been associated with outbreaks of healthcare-associated illness (HAIs). One in every 20 patients in U.S. acute care hospitals acquire a healthcare-associated illness, leading to consequences such as elevated morbidity, mortality, and a decrease in quality of life. In the patient environment, MRSA can remain viable from hours to up to 14 days. METHODS: Environmental surface materials were evaluated as new and worn. Material composition and properties were assessed to evaluate surface integrity and the influence on the disinfection of MRSA. Inoculated materials were used to assess MRSA viability over time and the efficacy of a manufacturer's recommended cleaning and disinfection product compared to a novel disinfectant. RESULTS: Environmental surface materials respond differently in appearance and roughness, when mechanically worn. When measuring MRSA survival, at 24 hr, MRSA colony forming unit (CFU) counts were reduced on the copper sheet surface and solid surface with cupric oxide. By 72 hr, all MRSA counts were zero. Bleach and the novel disinfectant were equally effective at disinfecting MRSA from all surface types. CONCLUSIONS: This study highlights a gap in knowledge about the impact of type and wear of environmental surface materials used in healthcare environments on contamination with epidemiologically important organisms. In conclusion, environmental surface material wear, properties, and cleaning and disinfection efficacy are important factors to consider when addressing HAIs.

Engineered aluminum nanoparticle induces mitochondrial deformation and is predicated on cell phenotype.

The main role of mitochondria is to generate the energy necessary for the cell to survive and adapt to different environmental stresses. Energy demand varies depending on the phenotype of the cell. To efficiently meet metabolic demands, mitochondria require a specific proton homeostasis and defined membrane structures to facilitate adenosine triphosphate production. This homeostatic environment is constantly challenged as mitochondria are a major target for damage after exposure to environmental contaminants. Here we report changes in mitochondrial structure profiles in different cell types using electron microscopy in response to particle stress exposure in three different representative lung cell types. Endpoint analyses include nanoparticle intracellular uptake; quantitation of mitochondrial size, shape, and ultrastructure; and confirmation of autophagosome formation. Results show that low-dose aluminum nanoparticles exposure (1 ppm; 1 µg/mL; 1.6 × 1 0-7 µg/cell)) to primary and asthma cells incurred significant mitochondrial deformation and increases in mitophagy, while cancer cells exhibited only slight changes in mitochondrial morphology and an increase in lipid body formation. These results show low-dose aluminum nanoparticle exposure induces subtle changes in the mitochondria of specific lung cells that can be quantified with microscopy techniques. Furthermore, within the lung, cell type by the nature of origin (i.e. primary vs. cancer vs. asthma) dictates mitochondrial morphology, metabolic health, and the metabolic stress response of the cell.

Novel perspectives on stomatal impressions: Rapid and non-invasive surface characterization of plant leaves by scanning electron microscopy.

Scanning electron microscopy (SEM) is widely used to investigate the surface morphology, and physiological state of plant leaves. Conventionally used methods for sample preparation are invasive, irreversible, require skill and expensive equipment, and are time and labor consuming. This study demonstrates a method to obtain in vivo surface information of plant leaves by imaging replicas with SEM that is rapid and non-invasive. Dental putty was applied to the leaves for 5 minutes and then removed. Replicas were then imaged with SEM and compared to fresh leaves, and leaves that were processed conventionally by chemical fixation, dehydration and critical point drying. The surface structure of leaves was well preserved on the replicas. The outline of epidermal as well as guard cells could be clearly distinguished enabling determination of stomatal density. Comparison of the dimensions of guard cells revealed that replicas did not differ from fresh leaves, while conventional sample preparation induced strong shrinkage (-40% in length and -38% in width) of the cells when compared to guard cells on fresh leaves. Tilting the replicas enabled clear measurement of stomatal aperture dimensions. Summing up, the major advantages of this method are that it is inexpensive, non-toxic, simple to apply, can be performed in the field, and that results on stomatal density and in vivo stomatal dimensions in 3D can be obtained in a few minutes.