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.     

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.     

Real-time white light reflection confocal microscopy using a fibre-optic bundle

We describe a real-time white light reflection con-focal microscope incorporating an optical fibre bundle and characterise the optical performance of the bundle. The use of an incoherent light source enables us, for the first time, to present speckle-free endoscopic reflected light confocal images. The system has potential application for in vivo studies.     

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.     

Automated assessment of keratocyte density in clinical confocal microscopy of the corneal stroma

Cell density in the corneal stroma is typically determined by counting the number of bright objects, presumably keratocyte nuclei, in images from clinical confocal microscopy. We present a program that identifies bright objects and counts those that most likely represent cells. Selection variables were determined from 125 normal corneas with cell densities that had been assessed manually. The program was tested on 17 corneas of patients before and at several intervals to 5 years after laser in situ keratomileusis (LASIK) surgery. In these corneas, which showed a decrease in cell density after surgery, the program identified cells as well as human observers did.     

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.     

Three-dimensional image formation in confocal microscopy

Three-dimensional (3-D) imaging in confocal microscopes is considered in terms of 3-D transfer functions. This leads to an explanation of axial imaging properties. The axial response was observed in both object-scanning and beam-scanning microscopes and the influence of off-axis examination investigated. By simple processing of multi-detector signals, imaging in both the axial and transverse directions can be improved.     

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.     

Vital confocal microscopy in bone

We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the medial proximal tibial plateau of the rabbit, and special small, self-focussing objectives (dry 10/0.25, water immersion 20/0.45, and oil immersion 45/0.65 and 120/1.0) which mated and matched to the conical window entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tissue using two genera of confocal microscope: multiple aperture disc, tandem scanning, microscopes for observation in reflection, and video rate confocal laser scanning microscopes for recording, mainly in the fluorescence mode. The latter allowed the study of a variety of intravenously administered substances, including fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluorescent markers, and replace them into the circulation. Calcein and tetracycline bind to the mineral front in bone: this labelling was studied in progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation of the system used both confocal fluorescence and scanning electron microscopy methods in the study of retrieved implants. These studies showed that the subimplant cortical bone remodelled to a less compact structure with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, with the matrix containing large numbers of large cells: some of this tissue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide further confirmation of the general utility of high-scan speed confocal methodology in physiology.     

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.     

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.     

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.     

Fluorescence saturation in confocal microscopy

The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approached that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.     

The significance of 3-D transfer functions in confocal scanning microscopy

The three-dimensional (3-D) transfer function is a useful concept for describing image formation in confocal scanning microscopy. From it we can derive the corresponding 2-D transfer function for in-focus imaging. In confocal transmission this can be derived analytically. The 1-D transfer function for on-axis imaging, which can be expressed in an analytical form even for confocal fluorescence with differing wavelengths of excitation and fluorescence, can be derived from the 3-D transfer function. The 2-D transfer function for in-focus imaging in confocal fluorescence microscopy with a finite-sized detector is also presented, which is shown to exhibit sign changes and can therefore result in reversals of image contrast.     

Quantitative fluorescence in confocal microscopy

The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen-stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations. In conventional as well as confocal quantitative fluorescence microscopy the relationship between the integrated fluorescence intensity and the integrated absorbance shows a maximum. This is due to inner filtering and re-absorption of the excitation light and emission light respectively. In conventional quantitative fluorescence microscopy the relationship is influenced by the numerical aperture of the objective lens. Under confocal observation, as measured with the BIO-RAD MRC-500 Confocal Imaging System, no influence of the numerical aperture of the objective lens on the relationship between the integrated fluorescence intensity and the integrated absorbance could be observed.     

用于激光共聚焦显微镜的光谱扫描机构

随着生物医学技术的发展,组织样本经常被多种荧光标记物标记,需要通过光谱成像的方法区分出样本中不同的成分。本文在共聚焦显微镜基础上,介绍了一种由精密丝杠和步进电机控制的狭缝机构实现光谱成像的方法,讨论了狭缝缝片的具体设计和狭缝运动精度对光谱带宽和波长准确度的影响。     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.     

Real-time confocal imaging, during active air abrasion – substrate cutting

Air abrasion cutting, using particulates accelerated in a controlled compressed gas stream, is currently being re-evaluated as a precision tissue removal technique for dental cavity preparation. The minimal vibrations and heat generated during cutting commend the technique for use in the shaping of fragile or brittle materials that are vulnerable to vibrations and thermal stresses.
Traditional air abrasion studies have relied solely upon post-procedure imaging, and cutting process details have been inferred from the nature of the residual surface. In this paper, however, a real-time confocal microscopic imaging method is described, which for the first time has allowed prior target structure characterization with subsequent imaging of cutting interactions and substrate failure patterns. Using internally focusing long working distance Hill objective lenses, focusing deep to a protective microscope slide and adhesive interfaces, unhindered remote image sampling within the bulk of specimens such as tooth tissue, acrylic and brittle ceramics was possible.
Moreover, areas of active cutting and inactive regions were identified within air abraded cavities during their creation. The characteristics of the finished cut surfaces were demonstrated and confirmed the findings of previous SEM studies. The method allowed direct control over all the known variables influencing cutting with particulate streams.