The Role of Scanning Electron Microscopy in Ophthalmic Science

The eyeglobe is one of the classical domains of Scanning Electron Microscopy (SEM) in biology as it exposes several inner and outer surfaces. Both corneal and conjunctival epithelia towards the tear film as well as the corneal endothelial cells facing the anterior chamber may be accurately evaluated. The architecture of the angle and particularly the morphology of the Schlemm\u27s canal inner wall are clarified by SEM more than by TEM serial reconstruction. The surfaces of the iris and ciliary body, the zonula and the choroidal vessel arrangement are described in great detail. Three distinct types of membrane anchoring devices are demonstrated among the lens fibers. SEM impressively describes the retina, but it has not yet added any new information as to previous observations in a more conventional way. SEM plays a fundamental role in teaching ocular anatomy and physiology as it makes more comprehensive the interrelationships among different structures. In addition, it represents a proper structural approach for the clinician who is familiar with the three-dimensional observations obtained by means of biomicroscopy, ophthalmoscopy and fluorescein angiography. Therefore, SEM application should be further spread and possibly joined to immunocytochemistry, in order to obtain a more dynamic and functional analysis of the eye

Scanning Electron Microscopy Application in Clinical Research

Our personal experience on the application of scanning electron microscopy in cardiology, gastroenterology and ophthalmology is reviewed. SEM has not yet significantly contributed to myocardium pathology. However, in the near future, SEM could be a reliable technique to complete the information available from other sources. As to atherosclerosis, SEM allowed us to improve our knowledge of the early stages of the disease; some pathological features, not always detected by conventional morphological examinations, can be documented. An important contribution to gastrointestinal pathology was made by SEM investigations both in the staging of some important diseases (i.e., coeliac disease, peptic ulcer, Crohn\u27s disease, ulcerative colitis) and in the follow-up of mucosal changes during therapy. In the ophthalmological field,SEM provided three-dimensional new information to clinicians, who are familiar with the biomicroscopic images. Our experience in hematology is still limited. However, in the lost few years SEM joined to immunocytochemistry allowed us to characterize cell populations in several blood diseases. Some procedures of particular interest in the management of human bioptic specimens are stressed in order to get to a complete correlative microscopy. We conclude that continuous and simultaneous correlations have to be carried out between SEM and other methods and instruments available for morphological investigation

The Lectin-Gold Technique: An Overview of Applications to Pathological Problems

Lectins are proteins, mainly of vegetal origin, which recognize glycosidic residues with high specificity; for this property they have been used for many studies of molecular biology. The colloidal gold represents at present the most popular electron dense marker employed in immunocytochemistry, since it offers intrinsic and unique characteristics which are superior to those displayed by the other markers. The cytochemical method which utilizes the gold-labelled lectins takes advantages from both the two systems, in order to optimize the localization of the glycoconjugates. The present paper reviews both the technical aspects of the preparation of the lectin-gold complex and its application to some selected pathological problems. In particular, the papers concerning the eye and ear tissues, the urinary, reproductive, nervous and digestive systems and the blood cells are quoted

Scanning Electron Microscopy in the Study of Campylobacter Pylori Associated Gastritis

The close association between Campylobacter pylori (CP), gastritis and peptic ulcer is now well established. Moreover increasing evidence has been collected of a major etiological role of CP in type B chronic gastritis. For this reason, searching for CP is essential in all patients with upper gastrointestinal symptoms. Scanning electron microscopy (SEM) is a most reliable technique for studying the distribution of microorganisms and their relationship to the gastric mucosal surface. The aim of this paper is to compare SEM to other routine methods of detection for CP, such as Giemsa staining on histological sections and Urease Microtiter Test (MT) on fresh tissue and to investigate the surface morphology of gastric mucosa colonized by CP and to correlate it with the histopathological picture. Thirty-seven biopsies taken from the gastric body and the antrum of 22 patients were used for each type of determination. The different parameters were graded semiquantitatively. Histology showed a normal mucosa in 4 cases, chronic superficial gastritis in 12 and chronic atrophic gastritis in 21 cases. SEM was more sensitive than histology and Urease MT in detecting Campylobacter pylori. This is due to the patchy distribution of this bacterium on gastric mucosa. For this reason SEM should always be performed when routine tests are negative. The presence of CP correlated significantly (p \u3c 0.001, Spearman Rank Correlation Test) with the neutrophilic infiltrate, thus with the activity of the gastritis. The CP associated gastritis has no distinctive surface features other than the presence of the bacterium SEM morphology of surface gastric mucous cells suggests that CP does not damage the lining epithelium directly. Neutrophils and inflammatory mediators could be involved in the production of the mucosal lesions

Neurotrophic keratitis: Current challenges and future prospects

Neurotrophic keratitis (NK) is a degenerative corneal disease caused by damage of trigeminal corneal innervation, which leads to spontaneous epithelial breakdown and corneal ulceration. The impairment of corneal sensory innervation causes the reduction of both protective reflexes and trophic neuromodulators that are essential for the vitality, metabolism, and wound healing of ocular surface tissues. A wide range of ocular and systemic conditions, including herpetic keratitis, ocular chemical burns, corneal surgery, diabetes, multiple sclerosis, and neurosurgical procedures, can cause NK by damaging trigeminal innervation. Diagnosis of NK requires careful investigation of any ocular and systemic condition associated with the disease, complete ocular surface examination, and quantitative measurement of corneal sensitivity. The clinical stages of NK range from corneal epithelial alterations (stage 1) to persistent epithelial defect (stage 2) and ulcer (stage 3), which may progress to corneal perforation. Management of NK is based on clinical severity, and the aim of the therapy is to halt the progression of corneal damage and promote epithelial healing. Although several medical and surgical treatments have been proposed, no therapies are currently available to restore corneal sensitivity, and thus, NK remains difficult and challenging to treat. The purpose of this review is to summarize available evidence on the pathogenesis, diagnosis, and treatment of NK. Novel medical and surgical therapies including the topical administration of nerve growth factor and corneal neurotization are also described

Characterization of Mucus Glycoconjugates in Normal Human Conjunctiva by Lectins in Light Microscopy, Transmission and Scanning Electron Microscopy

Maintenance of tear film in normal conditions is dependent on 1) mucus layer integrity and 2) the presence and distribution of conjunctival epithelial cell microvilli. In the present work a new methodology has been developed to gain correlative information about microprojection assessment and mucus composition, from the same specimen, by Light Microscopy (LM), Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). We have characterized the glycosidic residues secreted by goblet cells in normal human conjunctiva, by means of four lectins (WGA, ConA, PNA and SBA), conjugated with FITC for LM and with colloidal gold for TEM and SEM. The cytochemical reactions were performed on histological sections of paraffin-embedded material and on semithin and ultrathin sections of both Epon embedded material directly processed for TEM and of blocks recovered from SEM and reprocessed for TEM. WGA, ConA, PNA and SBA receptors were found to be constituents of the mucus produced by goblet cells in human conjunctiva. The granules of the so-called second mucus system (SMS) cells were labelled mainly by WGA. A difference in the quality of glycoconjugates between goblet cells and SMS cells has been also demonstrated. Our results provide an improved method to evaluate alterations of tear film that occur in many conjunctival diseases

Ocular surface analysis in patients affected with rheumatic diseases

Objective. The international criteria for primary Sjogren's Syndrome (SS I) diagnosis (Vitali et al. 2002) include the Schirmer test I and vital dye staining as tests for ocular surface involvement, but diagnosis can be reached also when the item for ocular signs is not satisfied. The purpose of our study was to evaluate the ocular surface in patients with Sjogren's Syndrome, non- Sjogren's autoimmune diseases and Sicca Syndrome, to understand whether the SS I diagnosis can be targeted also on other tests related to the ocular surface status. Methods. Clinical and cytological data were collected from 122 patients: 40 patients had diagnosis of Primary Sjogren's Syndrome, 51 a non Sjogren's autoimmune disease and 31 had symptoms of dry eye. A validated questionnaire on symptoms was filled by each patient; clinical tests included: Schirmer test I, Jones test, Ferning test, Break Up Time, corneal aesthesiometry, tear clearance test, vital dye staining of the ocular surface, scraping and impression conjunctival cytology. Data were statistically evaluated by using SPSS software and Mann-Whitney analysis on unpaired data. Results. Data show that the subjective symptoms score, tear production, tear turnover, corneal sensitivity and ocular surface integrity are affected in SS I patients, with a statistically significant difference when matched to the other two groups. Conclusions. Our results suggest to enlarge the spectrum of ocular surface analysis, to support and orient a differential diagnosis among the autoimmune diseases

Blood-Based Treatments for Severe Dry Eye Disease: The Need of a Consensus

The use of blood-based eye drops as therapy for various diseases of the ocular surface has become increasingly popular in ophthalmic practice during recent years. The rationale for their use is based on the promotion of cellular proliferation and migration thanks to the supply of metabolically active substances, in particular growth factors. Blood-derived eye drops have been used for the treatment of several ocular surface disorders, such as dry eye disease, corneal ulcer, persistent epithelial defect, neurotrophic keratitis, ocular surface burn, recurrent corneal erosion, and limbal stem-cell deficiency. Both autologous (from patients themselves) and heterologous (from adult donors or from cord blood sampled at birth)-derived products exist, and each source has specific pros and cons. Despite an extensive literature, several issues are still under debate and the aim of this manuscript is to review the indications, preparation methods and storage, characterization of content, rationale for clinical outcomes, patient stratification, length of treatment, and rationale for repeated treatments at disease relapse. A rationale based on a "5 Ws and 2 Hs" protocol is proposed as a way of thinking, with the attempt to clarify Who, Why, When, Where, What, and How to use these treatment options

Longitudinal tear protein changes correlate with ocular chronic gvhd development in allogeneic hematopoietic stem cell transplant patients

Ocular graft-versus-host disease (oGVHD) is a manifestation of chronic GVHD, frequently occurring in patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed tear protein changes before and after allogeneic HSCT, and correlated their levels with the oGVHD development. This retrospective study included 102 patients, and data were recorded before the conditioning treatment, and after 3 to 6 months postoperatively. Tear protein analysis was performed with the Agilent-2100 Bioanalyzer on individual tears sampled by aspiration. Total protein (TP), Lysozyme-C (LYS-C), Lactoferrin (LACTO), Lipocalin-1 (LIPOC-1), Transferrin (TRANSF), Albumin (ALB), and Zinc-alpha-2-glycoprotein (ZAG-2) levels were retrieved and statistically analyzed. Following HSCT forty-three patients developed oGVHD. TP, LACTO, LYS-C, and ZAG-2 levels significantly decreased post-HSCT as compared to pre HSCT levels. In univariate analysis, TP, LACTO, and ZAG-2 decrease was associated with an increased development of oGVHD (OR = 4.49; 95% CI, 1.9 to 10.5; p < 0.001; OR = 3.08; 95% CI 1.3 to 7.6; p = 0.01; OR = 11.1; 95% CI 2.7 to 46.6; p < 0.001, respectively). TRANSF post-HSCT levels significantly increased (OR 15.7; 95% CI, 4.1 to 52.2; p = 0.0001). No pre-post-HSCT changes were shown in ALB and LIPOC-1 levels. Data suggest that TP content, LACTO, TRANSF, and ZAG-2 pre-post changes might be significant predictors of oGVHD development

Correlative Scanning Electron Microscopy in the Study of Human Gastric Mucosa

We studied two aspects of the human gastric mucosa: - the surface morphology of mucous cells, as viewed by scanning electron microscopy (SEM); - the glycosidic components of intracellular mucins, characterized by means of lectins. The latter were conjugated with fluorescein isothiocyanate and with colloidal gold-silver for the visualization of the reaction products in light microscopy (LM) and in SEM (backscattered mode) respectively. The surface morphology of mucous cells appears to be correlated to the secretory state.In gastric ulcers we found a prevalence of non-secreting cells. A decrease in glycosidic receptors for fucose-binding lectin and galactose-(l-3)-N-acetyl-galactosamine-binding lectin was also observed. This suggests the presence of an impaired mucus secretion which may play a role in the pathogenesis of gastric ulcer. Spiral bacteria, supposed to be aetiologically related to peptic ulcer and gastritis, were easily detected by SEM. Intestinal metaplasia defined complete in LM showed surface morphology and glycosidic components different from those of true intestinal mucosa. This implies the necessity of taking into account also these parameters when classifying this lesion. The same applies to polyps. Our data indicate that correlative SEM may contribute further information on the pathogenesis and pathology of gastric diseases