Laser and tandem scanning confocal microscopic studies of rabbit corneal wound healing

The process of corneal endothelial wound healing was studied using laser and tandem scanning confocal microscopy (LSCM and TSCM). Following transcorneal freeze (TCF) injury, rabbit corneas were observed using ex vivo LSCM and in vivo TSCM. LSCM revealed the intracellular actin filament organization which, stained with phalloidin-FITC, in migrating endothelial cells, transformed fibroblast-like cells, stroma keratocytes, and epithelial cells during wound healing in corneal tissue. The TSCM provided sequential spatial observation of morphologic changes from endothelium to epithelium of the cornea during in vivo cellular repair of wound healing noninvasively on the same cornea without animal sacrifice. Ex vivo LSCM supported the morphologic analysis of the in vivo TSCM observations.     

The use of confocal microscopy in evaluating corneal wound healing after excimer laser keratectomy

Corneal wound healing following excimer laser keratectomy is the major cause of regression of treatment results. The amount of anterior strorhal haze that develops may be influenced by topical medications. Over a period of 6 months, we followed 15 New Zealand white rabbit eyes that underwent excimer laser keratectomy with the VISX 193-nm ArF laser at a fluence of 150 mJ/cm² for a depth of 130 μm. Eyes were randomized to treatment with prednisolone acetate, diclofenac sodium (Voltaren), a combination of both, and a control group. Drops were administered four times a day for 1 week, two times a day for 3 weeks, and the drops were then tapered. All eyes were reepithelialized by 5 to 7 days. The tandem scanning confocal microscope (TSCM) was used to evaluate the corneal wound in vivo weekly for a month and monthly for 6 months. During the early postoperative period, the TSCM revealed significant anterior stromal keratocyte activation with cell elongation and the spindle-shaped appearance of fibroblasts in all groups. Collagenous stromal scarring was evident initially, then slowly decreased in all treatment groups. This study shows that TSCM is clinically useful for successive in vivo examinations of corneal wounds after excimer laser keratectomy and for comparing the effects of various topical medications.     

Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: System design and calibration

A new depth encoding system (DES) is presented, which makes it possible to calculate, display, and record the z-axis position continuously during in vivo imaging using tandem scanning confocal microscopy (TSCM). In order to verify the accuracy of the DES for calculating the position of the focal plane in the cornea both in vitro and in vivo, we compared TSCM measurements of corneal thickness to measurements made using an ultrasonic pachymeter (UP, a standard clinical instrument) in both enucleated rabbit, cat, and human eyes (n = 15), and in human patients (n = 7). Very close agreement was found between the UP and TSCM measurements in enucleated eyes; the mean percent difference was 0.50 ± 2.58% (mean ± SD, not significant). A significant correlation (R=0.995, n=15, p< 0.01) was found between UP and TSCM measurements. These results verify that the theoretical equation for calculating focal depth provided by the TSCM manufacturer is accurate for corneal imaging. Similarly, close agreement was found between the in vivo UP and TSCM measurements; the mean percent difference was 1.67 ± 1.38% (not significant), confirming that z-axis drift can be minimized with proper applanation of the objective. These results confirm the accuracy of the DES for imaging of the cornea both ex vivo and in vivo. This system should be of great utility for applications where quantitation of the three-dimensional location of cellular structures is needed.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.     

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with aprobe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.     

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.     

Observation of the retina using the tandem scanning confocal microscope

A tandem scanning confocal microscope (TSCM) is currently being used to obtain high-resolution images of the human cornea in vivo. Advantages of confocal microscopy for in vivo imaging include optical sectioning and increased contrast through removal of scattered light. We have adapted the TSCM to view the retina in vivo by constructing an applanating lens and fitting the microscope with an imaging-intensifying camera of increased sensitivity. The microscope uses a spinning disc with 40,000 holes, each of 30 microns diameter, and a 100 W mercury arc lamp light source with a 455 nm long pass filter. The applanating lens is composed of three elements, two of which are movable for focusing. Images of a rabbit retina were obtained in vivo revealing the nerve fiber layer and blood vessels around the optic disc. The power density at the retina was calculated to be 3 mW/cm², which is well below the power levels of a direct or indirect ophthalmoscope. Magnification of the retinal image was approximately 60x and a 1 mm wide area of retina was in view. This prototype TSCM system demonstrates that images of a retina in vivo are obtainable with confocal microscopy and that the sharpness is comparable to standard fundus camera photography. Further modifications to improve the light level and alterations in the design of the objective should improve the quality of the images obtained and achieve the enhanced resolution of which, in theory, the confocal microscope is capable.     

Wide field scanning slit in vivo confocal microscopy of flattening-induced corneal bands and ridges

We used a wide field scanning slit confocal microscope to examine the response of the in vivo human cornea to flattening. Flattening-induced effects consisted of (1) anterior corneal mosaic, which appeared as a meshwork of intersecting stromal and Bowman's layer bands with overlying epithelial ridges; (2) deep and middle stromal bands, which were narrower than and unrelated in position to the anterior corneal mosaic; and (3) posterior surface ridges. The posterior surface ridges projected posteriorly into the anterior chamber consisted of endothelium, Descemet's membrane, and posterior stroma, and were unrelated in position to posterior stromal bands. Confocal microscopy is a promising modality in the examination of the cornea and its response to mechanical stress.     

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy

Objective: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods: Cardiac H9c2 cells were exposed to H₂O₂ (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H₂DCFDA). Results: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H₂O₂ compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H₂DCFDA. Conclusions: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013. © 2013 Wiley Periodicals, Inc.     

浅谈激光共聚焦显微镜使用技巧

本文简要介绍了激光扫描共聚焦（LSCM）成像原理,并以花粉为例,详细介绍了共聚焦针孔直径、光电倍增管检测器增益、激光强度、扫描速度、扫描方式、Z轴步距等重要参数设置对共聚焦成像的不同影响。探讨了正确使用LSCM的方法与技巧,如获取高质量的图像、图像保存及图像处理,以便为科技人员利用LSCM开展更多植物学与环境科学相关的研究提供参考。     

浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

Quantifying Microwear on Experimental Mistassini Quartzite Scrapers: Preliminary Results of Exploratory Research Using LSCM and Scale‐Sensitive Fractal Analysis

Although previous use‐wear studies involving quartz and quartzite have been undertaken by archaeologists, these are comparatively few in number. Moreover, there has been relatively little effort to quantify use‐wear on stone tools made from quartzite. The purpose of this article is to determine the effectiveness of a measurement system, laser scanning confocal microscopy (LSCM), to document the surface roughness or texture of experimental Mistassini quartzite scrapers used on two different contact materials (fresh and dry deer hide). As in previous studies using LSCM on chert, flint, and obsidian, this exploratory study incorporates a mathematical algorithm that permits the discrimination of surface roughness based on comparisons at multiple scales. Specifically, we employ measures of relative area (RelA) coupled with the F‐test to discriminate used from unused stone tool surfaces, as well as surfaces of quartzite scrapers used on dry and fresh deer hide. Our results further demonstrate the effect of raw material variation on use‐wear formation and its documentation using LSCM and RelA. SCANNING 35:28‐39, 2013. © 2012 Wiley Periodicals, Inc.     

Combination of multiphoton and reflective confocal imaging of cornea

We combine reflective confocal microscopy with multiphoton microscopy to form a minimally invasive technique to observe the cornea. The two imaging modalities allow detection of complementary information from the cornea. The autofluorescence signal shows the cytoplasm of epithelial cells, and the second harmonic generation signal is used to detect collagen, found mostly in the stroma of the cornea. The reflective confocal imaging allows detection of epithelial cells and keratocytes in the stroma. The system is first tested on bovine cornea. Assessment of the result on the bovine eye will be used to evaluate the potential of the system as a technique for in vivo clinical application.     

Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope

A small diameter (600 µm) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non‐focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM–probe combination is an improvement over conventional fluorescence–probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.     

Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.     

Scanning electron microscopy and morphometric analysis of superficial corneal epithelial cells in dromedary camel (Camelus dromedarius)

It is likely that superficial corneal epithelial cells (SCECs) of the dromedary camels have a significant role in their survival at arid and semiarid regions. To the best of our knowledge, SCECs of camels' eyes have not been characterized previously using scanning electron microscopy (SEM), combined with morphometric analysis. Therefore, in the current study, we aim to describe the shape, topographical distribution, and density of SCECs associated with morphometric analysis using SEM. Twelve healthy adult camels' corneas were obtained immediately after slaughter. Each cornea has been divided into nine parts: central (C), middle dorsal (MD), middle ventral (MV), middle nasal (MN), middle temporal (MT), peripheral dorsal (PD), peripheral ventral (PV), peripheral nasal (PN), and peripheral temporal (PT). SCECs were distinguished and characterized into light, medium, and dark mosaics. The polygonal cells have been externally covered with microplicae that were more numerous above the light cells. The topographic distribution of light, medium, and dark cells revealed a well-defined concentration of light cells in excess of other cells in all parts as follows: PV (92.5%), PN (78.5%), MN (78%), MT (74.7%), PD (73.8%), PT (70.7%), MV (68.7%), MD (66.3%), and C (19.3%). The PV part recorded the highest density of light cells, while the C portion showed the lowest density for the same cells. We concluded that the light cells extensively predominate in all parts of the camels' cornea except the C part, indicating an adaptive modification to the harsh environment. Additionally, the PV and PN parts represent the permanent and endogenous source as well as a proliferative reserve for SCECs in dromedary camel.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell-substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell–substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

Three-dimensional imaging of corneal cells using in vivo confocal microscopy

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real-time tandem scanning confocal microscopy has previously been used to generate high-magnification two-dimensional (2-D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3-D datasets. The development of a new objective lens, used in combination with specialized real-time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3-D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3-D structures and analysing 4-D phenomena at the cellular level in living animals. Three-dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.