Transcriptomic analysis of hub genes regulating albinism in light- and temperature-sensitive albino tea cultivars 'Zhonghuang 1' and 'Zhonghuang 2'.

Albino tea cultivars have high economic value because their young leaves contain enhanced free amino acids that improve the quality and properties of tea. Zhonghuang 1 (ZH1) and Zhonghuang 2 (ZH2) are two such cultivars widely planted in China; however, the environmental factors and molecular mechanisms regulating their yellow-leaf phenotype remain unclear. In this study, we demonstrated that both ZH1 and ZH2 are light- and temperature-sensitive. Under natural sunlight and low-temperature conditions, their young shoots were yellow with decreased chlorophyll and an abnormal chloroplast ultrastructure. Conversely, young shoots were green with increased chlorophyll and a normal chloroplast ultrastructure under shading and high-temperature conditions. RNA-seq analysis was performed for high light and low light conditions, and pairwise comparisons identified genes exhibiting different light responses between albino and green-leaf cultivars, including transcription factors, cytochrome P450 genes, and heat shock proteins. Weighted gene coexpression network analyses of RNA-seq data identified the modules related to chlorophyll differences between cultivars. Genes involved in chloroplast biogenesis and development, light signaling, and JA biosynthesis and signaling were typically downregulated in albino cultivars, accompanied by a decrease in JA-ILE content in ZH2 during the albino period. Furthermore, we identified the hub genes that may regulate the yellow-leaf phenotype of ZH1 and ZH2, including CsGDC1, CsALB4, CsGUN4, and a TPR gene (TEA010575.1), which were related to chloroplast biogenesis. This study provides new insights into the molecular mechanisms underlying leaf color formation in albino tea cultivars.

Recent advances and biotechnological applications of RNA metabolism in plant chloroplasts and mitochondria.

The chloroplast and mitochondrion are semi-autonomous organelles that play essential roles in cell function. These two organelles are embellished with prokaryotic remnants and contain many new features emerging from the co-evolution of organelles and the nucleus. A typical plant chloroplast or mitochondrion genome encodes less than 100 genes, and the regulation of these genes' expression is remarkably complex. The regulation of chloroplast and mitochondrion gene expression can be achieved at multiple levels during development and in response to environmental cues, in which, RNA metabolism, including: RNA transcription, processing, translation, and degradation, plays an important role. RNA metabolism in plant chloroplasts and mitochondria combines bacterial-like traits with novel features evolved in the host cell and is regulated by a large number of nucleus-encoded proteins. Among these, pentatricopeptide repeat (PPR) proteins are deeply involved in multiple aspects of the RNA metabolism of organellar genes. Research over the past decades has revealed new insights into different RNA metabolic events in plant organelles, such as the composition of chloroplast and mitochondrion RNA editosomes. We summarize and discuss the most recent knowledge and biotechnological implications of various RNA metabolism processes in plant chloroplasts and mitochondria, with a focus on the nucleus-encoded factors supporting them, to gain a deeper understanding of the function and evolution of these two organelles in plant cells. Furthermore, a better understanding of the role of nucleus-encoded factors in chloroplast and mitochondrion RNA metabolism will motivate future studies on manipulating the plant gene expression machinery with engineered nucleus-encoded factors.

Comparative transcriptomics provides novel insights into the mechanisms of selenium accumulation and transportation in tea cultivars (Camellia sinensis (L.) O. Kuntze).

Tea plants (Camellia sinensis) show discrepancies in selenium accumulation and transportation, the molecular mechanisms of which are not well understood. Hence, we aimed to conduct a systematic investigation of selenium accumulation and transportation mechanisms in different tea cultivars via transcriptome analysis. The Na2SeO3 and Na2SeO4 treatments improved selenium contents in the roots and leaves of three tea cultivars. The high selenium-enrichment ability (HSe) tea cultivars accumulated higher selenium contents in the leaves than did the low selenium-enrichment ability (LSe) tea cultivars. Transcriptome analysis revealed that differentially expressed genes (DEGs) under the Na2SeO3 and Na2SeO4 treatments were enriched in flavonoid biosynthesis in leaves. DEGs under the Na2SeO3 treatment were enriched in glutathione metabolism in the HSe tea cultivar roots compared to those of the LSe tea cultivar. More transporters and transcription factors involved in improving selenium accumulation and transportation were identified in the HSe tea cultivars under the Na2SeO3 treatment than in the Na2SeO4 treatment. In the HSe tea cultivar roots, the expression of sulfate transporter 1;2 (SULTR1;2) and SULTR3;4 increased in response to Na2SeO4 exposure. In contrast, ATP-binding cassette transporter genes (ABCs), glutathione S-transferase genes (GSTs), phosphate transporter 1;3 (PHT1;3), nitrate transporter 1 (NRT1), and 34 transcription factors were upregulated in the presence of Na2SeO3. In the HSe tea cultivar leaves, ATP-binding cassette subfamily B member 11 (ABCB11) and 14 transcription factors were upregulated under the Na2SeO3 treatment. Among them, WRKY75 was explored as a potential transcription factor that regulated the accumulation of Na2SeO3 in the roots of HSe tea cultivars. This study preliminary clarified the mechanism of selenium accumulation and transportation in tea cultivars, and the findings have important theoretical significance for the breeding and cultivation of selenium-enriched tea cultivars.

Transcriptome Analysis Reveals That Ascorbic Acid Treatment Enhances the Cold Tolerance of Tea Plants through Cell Wall Remodeling.

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid's protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea.

The role of ethylene in plant temperature stress response.

Temperature influences the seasonal growth and geographical distribution of plants. Heat or cold stress occur when temperatures exceed or fall below the physiological optimum ranges, resulting in detrimental and irreversible damage to plant growth, development, and yield. Ethylene is a gaseous phytohormone with an important role in plant development and multiple stress responses. Recent studies have shown that, in many plant species, both heat and cold stress affect ethylene biosynthesis and signaling pathways. In this review, we summarize recent advances in understanding the role of ethylene in plant temperature stress responses and its crosstalk with other phytohormones. We also discuss potential strategies and knowledge gaps that need to be adopted and filled to develop temperature stress-tolerant crops by optimizing ethylene response.

Two quinoline-based two-photon fluorescent probes for imaging of viscosity in subcellular organelles of living HeLa cells.

Two viscosity-sensitive two-photon fluorescent probes (QL and QLS) were designed and synthesized, which can be localized in lysosome and mitochondria in living HeLa cells, respectively. As the increases of viscosity from 2.55 to 1150 cP, the fluorescence quantum yield (Φ) of QL and QLS was increased by 28-fold and 37-fold, respectively. At the same time, its effective two-photon absorption cross section (ΦδTPA) was enhanced by 15-fold and 16-fold, respectively. Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with QL and QLS, revealed that lysosomal viscosity ranged from 100.76 to 254.74 cP and mitochondrial viscosity ranged from 92.21 to 286.79 cP. This type of fluorescent probe is helpful in the design and application of materials for monitoring diseases associated with abnormal viscosity.

Dual-state emission difluoroboron derivatives for selective detection of picric acid and reversible acid/base fluorescence switching.

A novel difluoroboron derivative (TPEBF) containing α-cyanostilbene and tetraphenylethylene units has been designed and synthesized. TPEBF emits strong fluorescence both in dilute solutions (ΦFL = 19.3% in THF) and in the solid state (ΦFL = 49.3%), which is significantly distinct from the case of the aggregation-caused quenching (ACQ) and aggregation-induced emission (AIE) chromophores. The dual-state emission properties of the compound overcome the limitation of single-state luminescence and enable it to be used in both solid and solution states. TPEBF with strong emission in solution is utilized for sensing picric acid (PA) with high selectivity and sensitivity in THF (LOD = 497 nM) and aqueous media (LOD = 355 nM). The mechanism was described for the synergy of fluorescence resonance energy transfer (FRET) and photoinduced energy transfer (PET) based on the UV-vis absorption and fluorescence spectra, 1H NMR and theoretical calculations results. On the other hand, the highly efficient emission in the solid state allows the compound to be cast on paper to switch external acid/base stimuli.

Two interacting ethylene response factors regulate heat stress response.

The ethylene response factor (ERF) transcription factors are integral components of environmental stress signaling cascades, regulating a wide variety of downstream genes related to stress responses and plant development. However, the mechanisms by which ERF genes regulate the heat stress response are not well understood. Here, we uncover the positive role of ethylene signaling, ERF95 and ERF97 in basal thermotolerance of Arabidopsis thaliana. We demonstrate that ethylene signaling-defective mutants exhibit compromised basal thermotolerance, whereas plants with constitutively activated ethylene response show enhanced basal thermotolerance. EIN3 physically binds to the promoters of ERF95 and ERF97. Ectopic constitutive expression of ERF95 or ERF97 increases the basal thermotolerance of plants. In contrast, erf95 erf96 erf97 erf98 quadruple mutants exhibit decreased basal thermotolerance. ERF95 and ERF97 genetically function downstream of EIN3. ERF95 can physically interact with ERF97, and this interaction is heat inducible. ERF95 and ERF97 regulate a common set of target genes, including known heat-responsive genes and directly bind to the promoter of HSFA2. Thus, our study reveals that the EIN3-ERF95/ERF97-HSFA2 transcriptional cascade may play an important role in the heat stress response, thereby establishing a connection between ethylene and its downstream regulation in basal thermotolerance of plants.

Preparation and biological evaluation of a novel agarose-grafting-hyaluronan scaffold for accelerated wound regeneration.

At present, seeking an effective dressing for wound regeneration has drawn considerable interest. In this paper, a novel agarose-grafting-hyaluronan (Ag-g-HA) scaffold was synthesized for rapid wound healing. Elemental analysis results showed that the HA grafting rate of Ag-g-HA was ∼69%. Ag-g-HA remained bioactive to accelerate cell proliferation and stimulate secretion of TNF-α for macrophagocyte RAW 264.7, and collagen I and collagen III for fibroblast 3T3. An i n vivo study demonstrated that Ag-g-HA showed a faster repair cycle and a better skin histological structure for a full-thickness skin defect. The collagen I, collagen III and TNF-α secreted by mice for Ag-g-HA were similiar to HA. Ag-g-HA showed a similiar biological activity to HA but had a longer degradation time through its improved insolubility. These findings demonstrate that the Ag-g-HA scaffold accelerated wound healing, and could be a promising novel scaffold for tissue engineering and regenerative medicine.

Discrepancies in Endothelial Cell Density Values of Human Donor Corneas Resulting From Comparison Between Specular Microscopes and Endothelial Analysis Methods.

PURPOSE: The methods for specular microscopy evaluation across eye banks differ, which may result in variability in endothelial cell density (ECD) values that influence the surgeon's decision about donor tissue. A comparison of instruments and analysis methods is conducted in this study. METHODS: Specular images were captured from 97 donor corneas using both HAI and Konan specular microscopes. A single best quality image of each cornea from each instrument was graded using the respective inherent software and analysis method (HAI: variable frame method; Konan: center method). All raw specular images were standardized for dimensions and regraded in the CellChek system in a blinded fashion. The grading variances and paired t test were performed between instruments in both inherent and standardized analyses. Correlation and Bland-Altman analyses between instruments were also performed. RESULTS: Using the software inherent within HAI and Konan, the mean ECD readings for the 97 corneas were 2764 ± 583 and 2605 ± 517 cell/mm (P = 0.045), respectively, with a variance of 8.05% (range 0.26%-27.2%). HAI resulted in a higher ECD value in 79 corneas (81.4%). In CellChek software analysis, the mean ECD readings did not differ (2609 ± 514 and 2496 ± 507 cells/mm, respectively, P = 0.127), with a variance of 5.6% (range 0.24%-19.8%). CONCLUSIONS: There is a slight statistically significant mean difference between the ECD values obtained from the 2 specular microscopes, which is negated by standardization to a single analysis method. Eye banks and surgeons should use caution in making decisions based only on very small differences in ECD between otherwise equivalent corneal donor tissues.

Unraveling the Linkage between Retrograde Signaling and RNA Metabolism in Plants.

Retrograde signals are signals that originate in organelles to regulate nuclear gene expression. In plant cells, retrograde signaling from both chloroplasts and mitochondria is essential for plant development and growth. Over the past few years, substantial progress has been made in unraveling the linkages between chloroplast retrograde signaling and nuclear RNA metabolism processes or plastidial RNA editing. These findings add to the complexity of the regulation of organelle-to-nucleus communication. Chloroplast development and function rely on the coordinated regulation of chloroplast and nuclear gene expression, especially under stress conditions. A better understanding of retrograde signaling and RNA metabolism, as well as their connection, is essential for breeding stress-tolerant plants to cope with the dynamic and rapidly changing environment.

The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress.

The dynamic and specific transcriptome for high light (HL) stress in plants is poorly understood because heat has confounded previous studies. Here, we perform an in-depth temporal responsive transcriptome analysis and identify the core HL-responsive genes. By eliminating the effect of heat, we uncover a set of genes specifically regulated by high-intensity light-driven signaling. We find that 79% of HL-responsive genes restore their expression to baseline within a 14-h recovery period. Our study reveals that plants respond to HL through dynamic regulation of hormones, particularly abscisic acid (ABA), photosynthesis, and phenylpropanoid pathway genes. Blue/UV-A photoreceptors and phytochrome-interacting factor (PIF) genes are also responsive to HL. We further show that ABA biosynthesis-defective mutant nced3nced5, as well as pif4, pif5, pif4,5, and pif1,3,4,5 mutants, are hypersensitive to HL. Our study presents the dynamic and specific high-intensity light-driven transcriptional landscape in plants during HL stress.

GSNOR provides plant tolerance to iron toxicity via preventing iron-dependent nitrosative and oxidative cytotoxicity.

Iron (Fe) is essential for life, but in excess can cause oxidative cytotoxicity through the generation of Fe-catalyzed reactive oxygen species. It is yet unknown which genes and mechanisms can provide Fe-toxicity tolerance. Here, we identify S-nitrosoglutathione-reductase (GSNOR) variants underlying a major quantitative locus for root tolerance to Fe-toxicity in Arabidopsis using genome-wide association studies and allelic complementation. These variants act largely through transcript level regulation. We further show that the elevated nitric oxide is essential for Fe-dependent redox toxicity. GSNOR maintains root meristem activity and prevents cell death via inhibiting Fe-dependent nitrosative and oxidative cytotoxicity. GSNOR is also required for root tolerance to Fe-toxicity throughout higher plants such as legumes and monocots, which exposes an opportunity to address crop production under high-Fe conditions using natural GSNOR variants. Overall, this study shows that genetic or chemical modulation of the nitric oxide pathway can broadly modify Fe-toxicity tolerance.

Representation of Central Endothelial Cell Density by Analysis of Single Best Specular Microscopy Image Regardless of Cell Size Variance.

PURPOSE: The purpose of this study is to evaluate whether a single best image can represent central endothelial cell density (ECD) in corneas of differing cell size coefficient of variance (CV). METHODS: Four hundred one healthy eyes but with variant CV values were enrolled. For each eye, three nonoverlapping central cornea endothelium images were obtained with Konan NSP-9900 specular microscope. ECD and CV were evaluated by two independent graders using the well-established Center method. Only corneas with high image quality rating (IQR) and ECD >800 cell/mm2 by both graders were included in the study. The study sample was stratified into five CV levels (CV ≤ 35; ≥36; ≥38; ≥40; and ≥45). In each CV level, the ECD agreement, ECD variance, and the correlation between the ECD variation and CV values were analyzed. In addition, the ECD intragrader reproducibility and interframe differences were also analyzed for all levels except CV ≤ 35. RESULTS: The study sample includes a total of 278 eyes. High ECD agreement for the two independent graders (intraclass correlation coefficient [ICC] > 0.99), high ECD intragrader reproducibility (ICC > 0.95), low ECD variance (2.0% ± 1.6%, overall), no correlation between the ECD variation and the CV value (P > 0.05), and no significant ECD difference among frames (P > 0.05) was found in any studied CV levels. CONCLUSIONS: CV does not appear to be associated with ECD variance in the central cornea. TRANSLATIONAL RELEVANCE: This finding highlights that in healthy corneas but with high CV values, ECD can be reliably analyzed using one single image of best quality.

GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling.

During development or under stress, chloroplasts generate signals that regulate the expression of a large number of nuclear genes, a process called retrograde signaling. GENOMES UNCOUPLED 1 (GUN1) is an important regulator of this pathway. In this study, we have discovered an unexpected role for GUN1 in plastid RNA editing, as gun1 mutations affect RNA-editing efficiency at multiple sites in plastids during retrograde signaling. GUN1 plays a direct role in RNA editing by physically interacting with MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2). MORF2 overexpression causes widespread RNA-editing changes and a strong genomes uncoupled (gun) molecular phenotype similar to gun1 MORF2 further interacts with RNA-editing site-specificity factors: ORGANELLE TRANSCRIPT PROCESSING 81 (OTP81), ORGANELLE TRANSCRIPT PROCESSING 84 (OTP84), and YELLOW SEEDLINGS 1 (YS1). We further show that otp81, otp84, and ys1 single mutants each exhibit a very weak gun phenotype, but combining the three mutations enhances the phenotype. Our study uncovers a role for GUN1 in the regulation of RNA-editing efficiency in damaged chloroplasts and suggests that MORF2 is involved in retrograde signaling.

Correlation between Guttata Severity and Thickness of Descemet's Membrane and the Central Cornea.

Purpose: To characterize and correlate guttata severity, Descemet's membrane thickness (DMT), central cornea thickness (CCT) in corneas with guttae using specular microscopy and spectral-domain optical coherence tomography (SD-OCT) and test the Doheny Image Reading Center (DIRC) specular microscopy-based corneal guttata severity scale. Methods: Forty-nine eyes of 49 patients with guttata and 36 eyes of age-matched of 36 normal controls were enrolled in the study. Three images of the central cornea and four of the peripheral cornea (inferior, superior, nasal and temporal) of each eye were taken using the Konan NSP-9900 specular microscope. A volume scan of the central cornea cross-section was collected on each eye using the Heidelberg Spectralis SD-OCT. The density of endothelial guttata based on specular images was graded on a 0-4 scale, and the Descemet's membrane thickness (DMT) and central corneal thickness (CCT) were manually measured by two trained graders. Results: The DIRC corneal guttata severity scale showed good reproducibility of all corneal endothelial images (weighted Kappa = 0.87). Mean DMT was 16.1 ± 2.4 µm in controls and 25.5 ± 10.9 µm in corneas with guttata (P < 0.001). Mean CCT was 552 ± 26 µm in controls and 603 ± 55 µm in corneas with guttata (P < 0.001). Guttata severity was significantly correlated with both DMT (r = 0.743, P < 0.001) and CCT (r = 0.569, P < 0.001). Age was moderately correlated with DMT (r = 0.472, P = 0.003) and mildly correlated with guttata severity (r = 0.285, P = 0.031), but was not correlated with CCT (r = 0.058, P = 0.681). Guttatta grade 3 corneas displayed an increase in DMT and guttata grade 4 was associated with a significant increase in CCT. Conclusions: The DMT and CCT are increased in corneas with guttata. The higher density of guttae is correlated with increased thickness. Specular microscopy combined with SD-OCT can be used as a good approach to assess the severity of FECD.

Analysis of ocular inflammation in anterior chamber-involving uveitis using swept-source anterior segment OCT.

PURPOSE: To evaluate the utility of swept-source (SS) optical coherence tomography (OCT) to objectively analyze the degree of anterior chamber (AC) inflammation. METHODS: Thirty-eight eyes of 32 patients with uveitis and 20 control eyes were enrolled. SS OCT B-scans were obtained, and the number of cells in the B-scans was counted using two methods: (1) manual grading by Point Picker plug-in of Image J ( http://bigwww.epfl.ch/thevenaz/pointpicker/ ) and (2) automated grading by the Image J Particle Analysis algorithm ( http://imagej.net/Particle_Analysis ). The automated and manual AC cell counts were correlated with the Standardization of Uveitis Nomenclature score. RESULTS: The average numbers of AC inflammatory cells counted by the automated method were 8 ± 4.0, 18 ± 3.0, 42 ± 14.0, 81 ± 32.0, 117 ± 57.0, and 275 ± 67.0 cells/mm2 for grades 0, 0.5 + , 1 + , 2 + , 3 + , and 4 + , respectively. For the same clinical categories, the average manual cell counts were 6 ± 4.0, 18 ± 3.0, 34 ± 14.0, 72 ± 32.0, 92 ± 43.0, and 168 ± 65.0 cells/mm2, respectively. Zero cells were detected in the AC of healthy eyes. The automated and manual methods were highly correlated (R = 0.98, p < 0.001) and showed good correlation with the clinical grading (R = 0.88, p < 0.001). A mean AC particle size of 117.4 ± 108.8 µm was obtained by the automated method. CONCLUSIONS: Quantification of the AC cells imaged by SS AS-OCT shows good correlation with categorical clinical severity assessments in uveitis eyes. This approach may provide a more objective method for monitoring uveitis and response to uveitis therapy.

Two AIEE-active α-cyanostilbene derivatives containing BF2 unit for detecting explosive picric acid in aqueous medium.

Two novel α-cyanostilbene derivatives bearing triphenylamine and BF2 groups are synthesized (named TPE-B and TPE-BN). The fluorescent emissions of compounds TPE-B and TPE-BN are hypochromatically shifted and bathochromically shifted, respectively, with increasing polarity of the solvents, suggesting that the two compounds have characteristic polarity-dependent solvatochromic effects. Furthermore, they show obvious aggregation-induced emission enhancement (AIEE) phenomenon in THF/water mixture solutions. Meanwhile, compounds TPE-B and TPE-BN emit orange and yellow fluorescence in their solid states, respectively. Most significantly, in aqueous medium, compounds TPE-B and TPE-BN can selectively and sensitively detect picric acid (PA) among a number of nitroaromatic compounds, and their limits of detection (LOD) are calculated as 1.26 × 10-6 M and 1.51 × 10-6 M, respectively. The recognition mechanism for PA can be attributed to the photo-induced electron transfer (PET) process and this is supported by density functional theory (DFT) calculation. This research provides two novel compounds for the rational design of AIEE-active materials for sensing systems.

Evaluation of the corneal epithelium in non-Sjögren's and Sjögren's dry eyes: an in vivo confocal microscopy study using HRT III RCM.

BACKGROUND: The corneal epithelium is directly affected in dry eye syndrome. Thus, we attempted to describe the morphological features and evaluate the cellular density within the corneal epithelial layers in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE) by in vivo confocal microscopy (IVCM). METHODS: Central cornea was prospectively imaged by IVCM in 68 clinically diagnosed aqueous tear-deficient dry eyes and 10 healthy age-matched control eyes. Morphological characteristics of corneal epithelial layers and cellular densities were evaluated by four trained graders from the Doheny Eye Institute. RESULTS: Corneal epithelium in dry eyes presents morphological changes such as areas of enlarged and irregular shaped cells. In comparison with controls, the density of superficial epithelial cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01); the density of the outer layer of wing cells was smaller but not significantly different in NSDE (P > 0.05), but was lower in the SSDE group (P < 0.01); the density of the inner layer of wing cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01) and the density of basal epithelial cells was lower in both the NSDE (P < 0.01) and SSDE groups (P = 0.01). For all cell counts, the interclass correlation coefficient showed good agreement between graders (ICC =0.75 to 0.93). CONCLUSIONS: IVCM represents a reliable technique for examining the corneal epithelial microstructural changes associated with dry eyes, as well as for objectively and reproducibly quantifying cell densities within all corneal epithelial layers.