Recent studies used impression cytology with scanning electron microscopy (SEM) to study the conjunctival surface of bovine eyes and normal human eyes. The purpose of this study was to evaluate the use impression cytology and SEM (ICSEM) in patients affected by tear film abnormalities. Forty-five patients were divided into three groups according to mild, moderate or severe subjective sensation of dry eye. Fifteen asymptomatic subjects served as control group. In all patients the tear film was evaluated with break-up time (BUT), Schirmer's, and Ferning test, whereas conjunctival epithelium was evaluated with impression cytology and optic microscopy (ICOM), and ICSEM. The Spearman rank correlation test was used to compare the outcome of these examinations with the subjective sensation of dry eye in each group, and to identify correlations among the five tests. ICSEM findings highly correlated with subjective dry eye sensation (Spearman correlation coefficient, 796; P<0.01). ICSEM revealed incipient epithelial damage (reduction or absence of microvilli) before the appearance of alterations of nucleus and cytoplasm of epithelial cells revealed by optic microscopy. The number of microvilli was correlated with the degree of tear film abnormalities and subjective sensation of dry eye (Spearman correlation coefficient, 796; P<0.01). ICSEM was very effective in detecting the reduction in the number of microvilli. Therefore, it could represent an effective method to detect alterations in the conjunctival epithelium resulting from tear film damage even before the epithelial damage occurs and is detected by optic microscopy.

The present study was concerned with the development of a new experimental model of dry eye using human reconstructed in vitro corneal epithelium (HCE). The model is based on the use of adapted culture conditions that induce relevant modifications at the cellular and molecular level thus mimicking dry eye. The HCE model was maintained in a controlled environmental setting (relative humidity <40% and 40 °C temperature) for 24 h and up to 72 h to induce dry eye. The evolution of the dry eye condition was assessed by histology, immunohistochemistry staining, scanning electron microscopy, and gene expression by using TaqMan gene assay technology (mucin-4 [MUC4], matrix metallopeptidase-9 [MMP9], tumor necrosis factor-α [TNF-α], and defensin β-2 [DEFB2). The effects of different commercially available tear substitutes on the induced dry eye condition were tested. This in vitro dry eye HCE model, that was well established within 24 h, has the characteristic features of a dry eye epithelium and could be satisfactorily used for preliminary assessment of the protective activity of some artificial tears. The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression. By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.

The common approach of the diagnosis of Alzheimer’s Disease (AD) is made with an analysis of the cerebrospinal fluid or the study of retinal fundus and the plaques formation through optical corneal tomography (OCT), or more simply with a fundus camera. Tears analysis is widely discussed in literature as an essential method to describe molecular and biochemical alterations in different diseases. The aim of our study was the identification with immunocytochemistry of Amyloid Beta-42 in tears from patients with or without familiarity for Alzheimer Disease, in order to make the diagnosis earlier and more accessible compared to other invasive methods. Our study was performed on tears from three phenotypically healthy subjects: two of them were Caucasian with Alzheimer familiarity (48 and 55 years old) and the other one was Asian without Alzheimer familiarity (45 years old) and affected by an adenoviral keratoconjunctivitis at the moment of withdrawal. Tear samples were collected from eye fornix and were examinated by immunocytochemistry (ICC) assay using anti-Amyloid Beta X-42 antibody. Two out of three tears samples showed positive Amyloid Beta-42. Considering that our patients were phenotypically healthy, the identification of Amyloid Beta-42 by ICC could be a candidable method to make the diagnosis of the disease earlier and more accessible and available then other current and invasive methods and it could be a candidate for a screening method too.

The authors performed an ex vivo and in vivo evaluation of the ultrastructural effects on the conjunctival epithelial cells of a new multiple-action tear substitute containing cross-linked hyaluronic acid, lipids and trehalose (Trimix^®), using scanning electron microscopy (SEM) with conjunctival impression cytology. The ex vivo study highlights the persistence and distribution of the product at 5 and 60 min on a monolayer of conjunctival epithelial cells and an increase in microvilli density at the 60 min evaluation. In vivo examination was conducted on three subjects with different grades of ocular surface inflammation, treated with one drop of the product twice daily for thirty days. At the baseline (T₀) and twelve hours after the last administration of the tear drop (T₃₀), impression cytology of the upper bulbar conjunctiva for SEM evaluation of conjunctival epithelial cells was carried out. Slit lamp examination (SLE), corneal and conjunctival Fluotest, tear film break-up time (TBUT), and ocular surface disease index (OSDI) questionnaires were also performed to correlate the ultrastructural results with the clinical findings. After 30 days of treatment, a significant improvement in all clinical and symptomatic parameters and in the condition of the ocular surface was detected, with microvillar regeneration and strengthening in all the patients, and a complete restoration in 2/3 of them. The persistence and distribution of the product on the epithelial cells was also noted 12 h after the last administration. The results, therefore, suggest a marked epitheliotropic effect along with a high residence time of the tear substitute.

Microvilli are bristle-like protuberances of the plasma membrane, which express the vitality of mucous and epithelial cells; their alteration indicates a condition of cellular suffering in a predictive sense, making it possible to establish how much an inflammatory state or toxic conditions affect cellular functionality. In this article, the authors evaluate the applications of scanning electron microscopy (SEM) examination to impression cytology (IC) of the bulbar conjunctiva for the assessment of microvillar alteration as an early ultrastructural indicator of ocular surface health. This method offers several advantages, starting with its simplicity: it involves the non-invasive application of a strip of bibulous paper to the bulbar or tarsal conjunctiva. Unlike conjunctival or corneal biopsies, which are surgical procedures, this technique is far less invasive and more comfortable for the patient. It also provides a more clinically relevant in vivo assessment compared to studies on cultured cell lines, which are mostly limited to scientific research and may not accurately reflect real-world conditions. This makes it an effective, repeatable, and patient-friendly option for detecting early pathological alterations of the ocular surface. It also represents a useful tool for evaluating the efficacy of topical drugs and the toxic effects of external factors and ophthalmic or systemic diseases. Finally, it allows for obtaining accessory information relating to goblet cells, the presence of inflammatory infiltrate, or any pathogens.

65 consecutive patients with presumed microbial keratitis and negative culture tests for bacteria and fungi obtained by corneal curettage were evaluated. All patients had undergone local broad spectrum antibiotic therapy for at least 5 days with no clinical improvement. After 48–72 h of wash-out they underwent scraping of the superior tarsal conjunctiva for cytological examination of cellular morphology in Scanning Electron Microscopy (SEM). The presence of pathogenic microorganisms was detected with this method in 62 of these patients, towards which specific therapy was carried out. Clinical improvement and eradication of microorganisms previously detected by SEM examination were observed in all positive patients over a time between 10 and 49 days. In three patients, no microorganisms were detected, but the presence of inflammatory cells (eosinophils and mast cells) or dry eye findings. This method could be useful to detect the presence of non-isolated microorganisms at common culture tests. The resolution of the infectious keratitis and the eradication of the pathogens at the subsequent cytological examination of cellular morphology in Scanning Electron Microscopy support the validity of the proposed method.