Content of clinker and other materials in personal thoracic aerosol samples from cement plants estimated by scanning electron microscopy and energy-dispersive X-ray microanalysis.

OBJECTIVES: To estimate the composition and exposure to clinker and other specific components in personal thoracic dust samples of cement production workers. METHODS: A procedure for the classification of airborne particles in cement production plants was developed based on classification trees. For this purpose, the chemical compositions of 27,217 particles in 29 material samples (clinker, limestone, gypsum, clay, quartz, bauxite, iron source, coal fly ash, and coal) were determined automatically by scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX). The concentrations of the major elements in cement (calcium, aluminium, silicon, iron, and sulphur) were used for the classifications. The split criteria of the classification trees obtained in the material samples were used to classify 44,176 particles in 34 personal thoracic aerosol samples. The contents of clinker and other materials were estimated, and the clinker contents were analysed statistically for differences between job types and job tasks. RESULTS: Between 64% and 88% of the particles from material samples were classified as actual materials. The material types with variable composition (clay, coal fly ash, and coal) were classified with the lowest consistency (64% to 67%), while materials with a more limited compositional variation (clinker, gypsum, and quartz) were classified more consistently (76% to 85%). The arithmetic mean (AM) of the clinker content in personal samples was 62.1%, the median was 55.3%, and 95% confidence interval (CI) was 42.6% to 68.1%. No significant differences were observed between job types. However, the clinker content in samples when workers handled materials with high clinker content was significantly higher than when materials with lower clinker content were handled, 85% versus 65% (P = 0.02). The limestone content was AM 14.8%, median 13.2% (95% CI 5.5 to 20.9), whereas the other materials were present with relative abundances of median ≤ 6.4%. DISCUSSION: Automated particle analysis by SEM-EDX followed by classification tree analysis quantified clinker with fairly high consistency when evaluated together with raw materials that are expected to be airborne in cement production plants. The clinker proportions for job types were similar. Tasks a priori ranked by assumed clinker content were significantly different and according to expectations, which supports the validity of the chosen methodology. CONCLUSIONS: The composition of personal samples of mineral aerosols in the cement production industry could be estimated by automated single particle analysis with SEM-EDX and classification by a classification tree procedure. Clinker was the major component in the thoracic aerosol that cement production workers were exposed to. Differences between job types were relatively small and not significant. The clinker content from tasks was in agreement with assumptions.

Analytic procedure for the evaluation of copper intermetallic diffusion in electroplated gold coatings with energy dispersive X-ray microanalysis.

A method for the determination of the intermetallic diffusion coefficient in the Cu-Au system is described based on energy dispersive X-ray techniques. XRF and EDS analysis were used to measure the thickness of the electroplated gold coating and the copper diffused through it, respectively. This information was used to obtain the diffusion coefficient through an equation based on Fick's law. Colour measurements and metallographic section analysis of the samples were also performed to evaluate alternative methods for a qualitative determination of diffusion rate. The thickness of the gold layer was chosen in agreement with what is used in decorative and functional applications (<1 µm). The measurements were performed on samples heated in a range of temperatures between 100 °C and 200 °C from 12 to 96 h. The results obtained follow a linear trend between the logarithm of the diffusion coefficient and the inverse of the temperature and are in line with the values found in the literature.

Chronic lip edema and pain secondary to lip augmentation procedure: histological, scanning electron microscopy and X-ray microanalysis evaluation.

BACKGROUND: In parallel with the increase in requests for filler injections, reported complications (immediate or delayed and transient or permanent) increased too. CASE REPORT: In the presented case report, a patient reported a delayed complication of filler injection. The complaints of the patient and the objective evaluation revealed the presence of multiple lip nodules, that were painful both spontaneously and at the touch, after 10 years from a lip filler injection. The surgical excision of the neoformations permitted the complete healing of the lesions. The histological analysis showed a not specific pattern, showing a material encapsulated by a tissue reaction, confirming that the possible cause of the multiple injuries could be attributable to the injection received years earlier. CONCLUSIONS: The professional who intends to perform filler injections needs a complete training process, both from a theoretical and practical point of view. Also, the patients must be informed of the possible risks associated with each product.

Synthesis and Structural Studies of New Selenium Derivatives Based on Covalent Functionalization of MWCNTs.

Modifying the surface of nanomaterials, such as carbon nanotubes, by introducing heteroatoms or larger functional groups into the structure causes a change in chemical properties-manifested in the increase in reactivity as well as a change in conductivity. This paper presents the new selenium derivatives obtained by a covalent functionalization of brominated multi-walled carbon nanotubes (MWCNTs). The synthesis was carried out in mild conditions (3 days at room temperature), and was additionally assisted with ultrasound. After a two-stage purification, the obtained products were identified and characterized by the following methods: scanning and transmission electron microscopy imaging (SEM and TEM), energy dispersive X-ray microanalysis (EDX), X-ray photoelectron spectroscopy (XPS), Raman and nuclear magnetic resonance (NMR), and X-ray diffraction (XRD). In the selenium derivatives of carbon nanotubes, the content of selenium and phosphorus reached 14 and 4.2 wt%, respectively.

Visual, spectral, and microchemical quantification of crystalline anomalies in otoliths of wild and cultured delta smelt.

Developmental abnormalities in otoliths can impact growth and survival in teleost fishes. Here, we quantified the frequency and severity of developmental anomalies in otoliths of delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish that is endemic to the San Francisco Estuary. Left-right asymmetry and anomalous crystalline polymorphs (i.e., vaterite) were quantified and compared between wild and cultured populations using digital image analysis. Visual estimates of vaterite were validated using X-ray diffraction, Raman spectroscopy, laser ablation ICPMS, and electron probe microanalysis. Results indicated that cultured delta smelt were 80 times more likely to contain a vateritic otolith and 18 times more likely to contain relatively large (≥ 15%) amounts of vaterite. Similarly, cultured fish exhibited 30% greater asymmetry than wild fish. These results indicate that cultured delta smelt exhibit a significantly higher frequency of vestibular abnormalities which are known to reduce fitness and survival. Such hatchery effects on otolith development could have important implications for captive culture practices and the supplementation of wild fish populations with cultured individuals.

Electron probe microanalysis of the elemental composition of phytoliths from woody bamboo species.

Electron probe microanalysis (EPMA) is promising for accurately determining elemental components in micro-areas of individual phytolith particles, interpreting compositional features and formation mechanisms of phytoliths in plants, identifying archeological and sedimental phytolith. However, the EPMA method of analyzing mounted slide phytoliths has not well been defined. In this study, we attempted different EPMA methods to determine the elemental compositions of phytoliths in mounted slides. Direct analysis of carbon (DAC) with other elements in phytolith could obtain abnormally high total values and carbon values. The method of carbon excluded in measuring elements (non-carbon analysis (NCA)) was feasible to obtain elemental compositions in phytolith. The NCA method was conducive to obtain the factual elemental compositions of an individual phytolith (morphotype) when the carbon content of phytolith was relatively low. The EPMA results of phytoliths from 20 bamboo species (three genera) showed that phytolith was dominantly composed of SiO2 but also included low contents of diverse other elements. The EPMA of phytoliths can provide the elemental composition of micro-areas of an individual phytolith particle. The elemental compositions of phytolith varied with their morphotypes, the genera and ecotype of bamboos. The EPMA of elemental compositions in phytoliths is a potential tool to study the formation mechanism of phytoliths, plant taxonomical identification, archaeological and paleoenvironmental reconstruction.

Improved Watershed Algorithm-Based Microscopic Images Combined with Meibomian Gland Microprobe in the Treatment of Demodectic Blepharitis.

The objective of the study was to explore microscopic images under a watershed segmentation algorithm combined with meibomian gland microprobe in the treatment of demodectic blepharitis. For segmenting the connected target objects in the image, the watershed algorithm was utilized first to obtain the target region in the image, and then, the fuzzy C-means (FCM) clustering algorithm was used to cluster the targets. The different grayscale regions in the microscopic images were segmented. 90 patients with demodectic blepharitis-related dry eyes were selected, and they were divided into experimental group 1 (group E1, n = 30), experimental group 2 (group E2, n = 30), and control group (group CG, n = 30). The breakup time (BUT) of the tear film, the subjective score of clinical symptoms, and the number of mites were compared among the three groups before and after treatment. The results showed that after treatment, the indicators of group E1 and group E2 were significantly lower than those before treatment, and the differences were statistically significant (P < 0.05). The treatment effect of group E1 was significantly better than that of the other two groups (P < 0.05). The subjective clinical symptom scores of groups E1, E2, and CG were 13.43 ± 1.41, 13.51 ± 1.41, and 13.64 ± 0.84, respectively, before treatment, and those after treatment were 3.1 ± 1.841, 5.4 ± 0.661, and 13.4 ± 0.841, respectively. The clinical sign scores of the groups E1 and E2 after treatment were remarkably different from those before treatment (P < 0.05). Compared with the scores of clinical signs and clinical symptoms after treatment, those of group E1 showed the largest differences, indicating the best treatment effect. In conclusion, the treatment effect of blepharitis could be promoted with the improved watershed algorithm, and the microscopic images combined with meibomian gland microprobe gave the better effect in the treatment of demodectic blepharitis than the conventional drug heat compress.

In Vivo Biocompatibility Investigation of an Injectable Calcium Carbonate (Vaterite) as a Bone Substitute including Compositional Analysis via SEM-EDX Technology.

Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.

Structure and mineralization of the spearing mantis shrimp (Stomatopoda; Lysiosquillina maculata) body and spike cuticles.

Stomatopoda is a crustacean order including sophisticated predators called spearing and smashing mantis shrimps that are separated from the well-studied Eumalacotraca since the Devonian. The spearing mantis shrimp has developed a spiky dactyl capable of impaling fishes or crustaceans in a fraction of second. In this high velocity hunting technique, the spikes undergo an intense mechanical constraint to which their exoskeleton (or cuticle) has to be adapted. To better understand the spike cuticle internal architecture and composition, electron microscopy, X-ray microanalysis and Raman spectroscopy were used on the spikes of 7 individuals (collected in French Polynesia and Indonesia), but also on parts of the body cuticle that have less mechanical stress to bear. In the body cuticle, several specificities linked to the group were found, allowing to determine the basic structure from which the spike cuticle has evolved. Results also highlighted that the body cuticle of mantis shrimps could be a model close to the ancestral arthropod cuticle by the aspect of its biological layers (epi- and procuticle including exo- and endocuticle) as well as by the Ca-carbonate/phosphate mineral content of these layers. In contrast, the spike cuticle exhibits a deeply modified organization in four functional regions overprinted on the biological layers. Each of them has specific fibre arrangement or mineral content (fluorapatite, ACP or phosphate-rich Ca-carbonate) and is thought to assume specific mechanical roles, conferring appropriate properties on the entire spike. These results agree with an evolution of smashing mantis shrimps from primitive stabbing/spearing shrimps, and thus also allowed a better understanding of the structural modifications described in previous studies on the dactyl club of smashing mantis shrimps.

The f-ratio model for quantitative X-ray microanalysis.

The f-ratio method is a new quantitative X-ray microanalysis method developed based on a cold field emission scanning electron microscope/energy dispersive spectroscopy system. The f-ratio is calculated with the characteristic X-ray intensities, and the Monte Carlo simulation is employed to build the theoretical relation between the system composition and the f-ratio. In this study, the f-ratio model is formulated with the elemental concentrations and the f-ratio coefficients. The f-ratio models in the binary S-Fe system and the ternary O-Al-Si system were studied, and the beam energy effects were investigated. The quantitative analyses were performed on the standard pyrite (FeS2) and kyanite (Al2SiO5) specimens, and the results show that the f-ratio model is able to achieve a satisfying accuracy.

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis.

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

An x-ray microanalytical method for measuring in vivo element and water concentrations, relating to osmoregulation, in cells and tissues of the posterior eye.

Osmoregulation is critical for cell and tissue survival yet there are relatively few methods available to determine osmotic gradients from water and elemental concentration either in single cells or across multiple cellular layers of tissue. X-ray microanalysis of frozen-hydrated preparations in a scanning electron microscope is one such powerful, sensitive, nondestructive technique. Here we use x-ray microanalysis to quantitatively analyse intracellular element concentrations and oxygen concentrations, as a proxy for water concentrations, in selected individual cells of the posterior eye. Using frozen-hydrated preparations of the retinal complex of chicken eyes, it is shown that structural preservation is sufficient to identify cell layers and individual cells. The quantitative analysis of selected areas in the photoreceptor layer, inner nuclear layer and ganglion cell layer, where specific cell types were known to be present, provided measurements of intracellular element concentrations comparable with the analysis of individual cells. It is also shown that in the cells of the retinal pigment epithelium and outer photoreceptor segments elemental analyses were reasonably consistent at the cellular level in different depth levels of the same sample. Comparison of oxygen concentrations, as a proxy for water concentration, at two accelerating voltages (15 and 5 kV) indicated that at 15 kV oxygen concentration was largely derived from intracellular water. Water concentrations could be calculated and concentrations of diffusible elements (Na, K) could be defined in mmol/L. From the latter it is possible to calculate osmotic concentrations of individual cells and osmotic gradients across the tissue. LAY DESCRIPTION: Understanding many cellular processes, in both healthy and diseased states, depends on knowing how the water content of cells and their surrounding fluids is controlled. The transport of water is generally down its concentration gradient or against the osmotic concentration gradient defined by solutes such as sodium, potassium and chloride dissolved in the water. We have refined a microanalytical method, that detects the x-rays emitted from specific elements when they are bombarded by electrons in a scanning electron microscope, to apply it to the analysis of the retina of the eye. The method facilitates the measurement of the elemental composition, water and osmotic concentration gradients of cells and tissues in the eye, that may be involved in the development of myopia, or short sightedness, a condition that afflicts many people including some 80 - 90% of children in Asia.

Fibronectin glomerulopathy: A case report and literature review / Glomerulopatía por fibronectina: informe de un caso y revisión de la literatura

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Root silicon deposition and its resultant reduction of sodium bypass flow is modulated by OsLsi1 and OsLsi2 in rice.

Silicon (Si) can alleviate salt stress by decreasing Na+ bypass flow in rice (Oryza sativa L.), however, the mechanisms underpinning remain veiled. In this study, we investigated the roles of OsLsi1 and OsLsi2 in Si-induced reduction of bypass flow and its resultant alleviation of salt stress by using lsi1 and lsi2 mutants (defective in OsLsi1 and OsLsi2, respectively) and their wild types (WTs). Under salt stress, Si promoted plant growth and decreased root-to-shoot Na+ translocation in WTs, but not in mutants. Simultaneously, quantitative estimation and fluorescent visualization of trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic (PTS, an apoplastic tracer) showed Si reduced bypass flow in WTs, but not in mutants. Energy-dispersive X-ray microanalysis (EDX) showed Si was deposited at root endodermis in WTs, but not in mutants. Moreover, results obtained from root split experiment using lsi1 WT showed down-regulated expression of Si transport genes (OsLsi1 and OsLsi2) in root accelerated Si deposition at root endodermis. In summary, our results reveal that Si deposition at root endodermis and its resultant reduction of Na+ bypass flow is modulated by OsLsi1 and OsLsi2 and regulated by the expression of OsLsi1 and OsLsi2, implying that root Si deposition could be an active and physiologically-regulated process in rice.

Multimodal X-ray microanalysis of a UFeO4: evidence for the environmental stability of ternary U(v) oxides from depleted uranium munitions testing.

An environmentally aged radioactive particle of UFeO4 recovered from soil contaminated with munitions depleted uranium (DU) was characterised by microbeam synchrotron X-ray analysis. Imaging of uranium speciation by spatially resolved X-ray diffraction (µ-XRD) and X-ray absorption spectroscopy (µ-XAS) was used to localise UFeO4 in the particle, which was coincident with a distribution of U(v). The U oxidation state was confirmed using X-ray Absorption Near Edge Structure (µ-XANES) spectroscopy as +4.9 ± 0.15. Le-Bail fitting of the particle powder XRD pattern confirmed the presence of UFeO4 and a minor alteration product identified as chernikovite (H3O)(UO2)(PO4)·3H2O. Refined unit cell parameters for UFeO4 were in good agreement with previously published values. Uranium-oxygen interatomic distances in the first co-ordination sphere were determined by fitting of Extended X-ray Absorption Fine Structure (µ-EXAFS) spectroscopy. The average first shell U-O distance was 2.148 ± 0.012 Å, corresponding to a U valence of +4.96 ± 0.13 using bond valence sum analysis. Using bond distances from the published structure of UFeO4, U and Fe bond valence sums were calculated as +5.00 and +2.83 respectively, supporting the spectroscopic analysis and confirming the presence of a U(v)/Fe(iii) pair. Overall this investigation provides important evidence for the stability of U(v) ternary oxides, in oxic, variably moist surface environment conditions for at least 25 years.

Quantitative toxicological study of dose-dependent arsenic-induced cells via synchrotron-based STXM and FTIR measurement.

Inorganic arsenic (iAs) is a well-known naturally occurring metalloid with abundant hazards to our environment, especially being a human carcinogen through arsenic-contaminated drinking water. The iAs-related contamination is usually examined by a chemical assay system or fluorescence staining technique to investigate iAs accumulation and its deleterious effects. In this work, we present a dual-modality measurement and quantitative analysis methods for the overall evaluation of various dose-dependent iAs-related cytotoxicological manifestations by the combination of the synchrotron-radiation-based scanning transmission soft X-ray microscopy (SR-STXM) and Fourier transform infrared micro-spectroscopy (SR-FTIR) techniques. The gray level co-occurrence matrix (GLCM) based machine learning was employed on SR-STXM data to quantify the cytomorphological feature changes and the dose-dependent iAs-induced feature classifications with increasing doses. The infrared spectral absorption peaks and changes of dose-dependent iAs-induced cells were obtained by the SR-FTIR technique and classified by the multi-spectral-variate principle component analysis (PCA-LDA) method, showing the separated spatial distribution of dose-dependent groups. In addition, the quantitative comparisons of trivalent and pentavalent iAs under high dose conditions (iAsIII_H & iAsV_H) demonstrated that iAsIII_H and its compounds were more toxic than iAsV_H. This method has a potential in providing the morphological and spectral characteristics evolution of the iAs-related cells or particles, revealing the actual risk of arsenic contamination and metabolism.

Power of Scanning Electron Microscopy and Energy Dispersive X-Ray Analysis in Rapid Microbial Detection and Identification at the Single Cell Level.

The demand for rapid, consistent and easy-to-use techniques for detecting and identifying pathogens in various areas, such as clinical diagnosis, the pharmaceutical industry, environmental science and food inspection, is very important. In this study, the reference strains of six food-borne pathogens, namely, Escherichia coli 0157: H7 ATCC 43890, Cronobacter sakazakii ATCC 29004, Salmonella Typhimurium ATCC 43971, Staphylococcus aureus KCCM 40050, Bacillus subtilis ATCC 14579, and Listeria monocytogenes ATCC 19115, were chosen for scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. In our study, the time-consuming sample preparation step for the microbial analysis under SEM was avoided, which makes this detection process notably rapid. Samples were loaded onto a 0.01-µm-thick silver (Ag) foil surface to avoid any charging effect. Two different excitation voltages, 10 kV and 5 kV, were used to determine the elemental information. Information obtained from SEM-EDX can distinguish individual single cells and detect viable and nonviable microorganisms. This work demonstrates that the combination of morphological and elemental information obtained from SEM-EDX analysis with the help of principal component analysis (PCA) enables the rapid identification of single microbial cells without following time-consuming microbiological cultivation methods.

Conjunctival tattoo with inadvertent ocular globe penetration and vitreous involvement: Clinico-pathological correlation and scanning electron microscopy X-ray microanalysis.

PURPOSE: The aim is to report a case of conjunctival tattooing with inadvertent injection of tattoo ink into the vitreous cavity and its consequences, the scanning electron microscopy X-ray microanalysis of the ink components, and the microscopic findings of the affected conjunctiva and vitreous. METHODS: Descriptive case report. RESULTS: A 32-year-old man complained of ocular pain and blurred vision after undergoing a subconjuctival red ink tattoo in his left eye. Ophthalmologic examination revealed best corrected visual acuity of 20/80 and intraocular pressure of 26 mmHg. Pain was elicited with eye movements. The bulbar conjunctiva was colored intense red. In the anterior chamber, pigment granules and filaments were suspended on the aqueous humor, and lens capsule was also stained red. Ultrasonography showed high-density non-mobile echoes in the conjunctiva; anterior chamber and vitreous cavity revealed high-density mobile echoes corresponding to pigment particles. Conjunctival tattoo with inadvertent globe penetration was the clinical diagnosis. The patient received medical and surgical treatment. Histopathological examination of the conjunctiva showed red pigment globular deposits within the stroma, and neutrophils and sparse histiocytes with similar intracytoplasmic pigment granules were seen. No granulomatous foreign body reaction was noticed. Vitreous material contained pigment granules; no inflammatory cells were observed. Scanning electron microscopy X-ray microanalysis of the tattoo red ink revealed significant signals of iron, barium, and copper. CONCLUSION: Conjunctival tattoo is a new form of body decoration gaining worldwide popularity. This procedure is performed by untrained professionals causing severe ocular complications including blindness. Safety regarding tattoo ink needs further study as the composition varies among colors. Strict regulations on this matter should be considered.

Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance.

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.