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Four-Dimensional Multiphoton Confocal Microscopy: The New Frontier in Cellular Imaging
Laboratory Science JAMES V. JESTER, PHD, SECTION EDITOR
Four-Dimensional Multiphoton Confocal Microscopy: The New Frontier in Cellular Imaging JAMES V. JESTER, PHD,1 BRANT R. WARD,2 AKIRA TAKASHIMA,PHD,2 JOEL GATLIN,PHD,3 J. VICTOR GARCIA,PHD,3 H. DWIGHT CAVANAGH, MD, PHD,1 AND W. MATTHEW PETROLL, PHD1 ABSTRACT This paper reviews new developments in microscopy that combine gene transfer technology, multiphoton confocal fluorescence microscopy, live cell imaging and digital imaging techniques that provide unique insights into the complex physiological processes involved in tissue function at the cellular and subcellular level. The evolution of this novel, new technology is discussed with particular attention to earlier achievements in noninvasive ocular surface imaging. The practical basis of confocal microscopy, multiphoton confocal fluorescence microscopy, and the vital fluorescent labeling of cells in living tissues are also discussed. Additionally, one application using retroviral gene transfer to express enhanced green fluorescent protein in living wound healing fibroblasts is presented as an example of how living biology can be studied in situ in four dimensions (x, y, z, time). KEYWORDS cornea, confocal microscopy, gene transfer, keratocyte
Accepted for publication December 2003. From the Departments of Ophthalmology,1 Dematology,2 and Internal Medicine3 at the University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. Supported in part by NIH grants NEI EY07348 and EY13215 and an unrestricted grant and Senior Scientist Award (JVJ) from Research to Prevent Blindness, Inc, New York. Presented in part at the Sunday Symposium, Ocular Wound Healing: A Symposium Dedicated to Dr. David Maurice, at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 4, 2003. The authors have no proprietary interest in any product or concept discussed in this article. Single copy reprint requests to James V. Jester, PhD (address below). Abbreviations are printed in boldface where they first appear with their definitions. Corresponding author: James V. Jester, PhD, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9057. Phone: (214) 648-7215. Fax: (214) 648-8447. Email: [email protected] ©2004 Ethis Communications, Inc. All rights reserved.
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I. INTRODUCTION t is generally recognized that normal tissue physiology and disease mechanisms involve complex and dynamic sets of interactions between cells, growth factors, cytokines, and an extracellular matrix environment that regulate downstream intracellular events controlling gene expression and cell function within tissues. While conventional cell and molecular approaches using tissue culture and biochemical sampling have dramatically increased our understanding of these events, this knowledge has come from ex vivo studies which separate, isolate and otherwise dissociate cells and tissues from their native environment. This reductionist approach has led to conflicting results when cellular and molecular responses from intact tissues are compared to those obtained from tissue culture models. In an attempt to return to the biology of the tissue and organism, recent tissue culture models have employed mixed cell cultures and three-dimensional extracellular matrix constructs to study the effects of cell-cell and cellmatrix interactions in controlling cell function in a more dynamic manner. For the most part, however, the totality of these interactions in the organism is best duplicated in the intact, living tissue, necessitating the development of better noninvasive approaches to studying cell function in living systems. Recent technological breakthroughs in imaging and gene transfer provide the opportunity to study the fourdimensional biology of tissues at the cellular and molecular level without the need to remove cells from their native environment. The application of this technology to the study of ocular surface physiology and disease is based on past achievements in the development of noninvasive ocular imaging combined with developments in multiphoton confocal fluorescence microscopy and viral vector technology. The purpose of this review is to familiarize the reader with the development of past and new technologies and to demonstrate how, through the combined application, we can begin to study the four-dimensional cell physiology in intact tissue systems. Specifically, this paper will first discuss past achievements of David Maurice, whose tireless interest in the biology of the eye moved him to
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FOUR-DIMENSIONAL IMAGING / Jester, et al OUTLINE I. Introduction II. Early developments in noninvasive ocular imaging III. Confocal microscopy A. History B. Principles and techniques C. Interpretation of images IV. Multiphoton fluorescence confocal microscopy A. Principles and techniques B. Advantages of multiphoton excitation V. Fluorescent labeling of living cells A. Limitations of current probes B. New labeling techniques VI. Four-demensional study of corneal wound healing VII. Future studies
develop new methods to observe ocular biology in situ, establishing him as an early pioneer in the development of noninvasive ocular imaging. Secondly, we will discuss the general area of confocal microscopy, followed by more recent developments in multiphoton confocal fluorescence microscopy that provide high resolution images of cells in living tissues. These, when combined with appropriate fluorescent probes, can be used to generate sequential, three- and four-dimensional images of cells and subcellular structures to study cell function. We will then present one approach that we have used to label cells in the live animal with a fluorescent marker to study four-dimensional cell biology in situ. Finally, we will briefly discuss how this type of approach can be used to direct future studies and answer important questions concerning corneal cell biology and development.
neal endothelium, further instrument refinement by Maurice2 and later by Koester3 produced a scanning slit microscope design that provided a wider field of view that promised images of not only the corneal endothelium but also the epithelium and stromal keratocytes. Many of these initial images obtained with the scanning slit microscope4 have been duplicated by recently developed in vivo confocal microscopes that use different designs that essentially replicate and extend Maurice’s original achievements using a thin scanning slit. Maurice was also the first to develop a functional artificial anterior chamber, and he established the conditions required for the maintenance of normal corneal endothelial function under ex vivo conditions.5,6 Combining the specular microscope with the Dikstein-Maurice chamber provided new insights into endothelial function and corneal transparency and represented the first studies of ocular surface tissue physiology at a cellular level. New methods in animal restraint devised by Maurice to facilitate continued sequential observation of the cornea7 and the recent application of vital staining of corneal cells using fluorescent dyes have led to the development of more dynamic observations of corneal cell biology and insights into the role of tears in the induction of keratocyte cell death.8 III. CONFOCAL MICROSCOPY A. History
Maurice’s early work set the stage for a dramatic expansion in our abilities to microscopically study the ocular surface in situ. Newer approaches based on the scanning slit originally designed by Maurice and the scanning pinhole as first designed by Petran et al using confocal
II. EARLY DEVELOPMENTS IN NONINVASIVE OCULAR IMAGING
David Maurice (1922–2003) was a pioneer in the design of noninvasive instrumentation to study the ocular surface and was the first to develop the “specular microscope” and to study the corneal endothelium in the living eye.1 Using a narrow slit beam for illumination and a divided objective aperture to separate the light reflected at the corneal surface from the weaker “specular” reflection of the corneal endothelium, he generated the first noninvasive optical microscopic sections through the thickness of the whole in vivo cornea. While the initial design of this microscope provided a narrow view of the cor-
Figure 1. Diagram of confocal microscope optics that collects light from the focal plane (A) and excludes scattered or excited light from the defocused plane (B).
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optics9,10 now provide high resolution images of the ocular surface and quantitative measurements of cell density,11,12 tissue thickness,13 and light scattering.14 For a more complete review of the development and design of the confocal microscope, the reader is referred to several excellent reviews that cover in more detail the technology and instrumentation that is currently available.15-17 Briefly, the confocal microscope was invented in 1957 by Minsky, who designed an optical microscope that focused illuminating light using lenses within a small volume of the specimen and then collected light from this same volume using similar lens to focus the light back to a detector.18 Since both the illumination side and the detection side of the microscope were in the same focus, the microscope was referred to as confocal. The theoretical applications of this approach have been formally developed and extended by Wilson and Sheppard19 and Sheppard and Cogswell.20 B. Principles and Techniques
Figure 2. Representative micrographs obtained from the mouse cornea using in vivo confocal microscopy (A and B) compared to laser scanning fluorescence confocal microscopy (C and D). Both represent three-dimensional data sets shown as an orthogonal cabinet projections (A) or as an XZ projection (C). Images shown in B and D represent single 2-dimensional slices taken from the corneal stroma. Arrow in A, indicates the interface between the corneal epithelium and stroma in the mouse. Fluorescent images are from rhodamine conjugated phalloidin (Red) and Syto 50 (Cyan) to repectively stain actin filaments and cell nuclei.
As demonstrated in Figure 1, current confocal microscopes essentially use a variable-sized illumination pinhole (Figure 1A). Light from the pinhole is directed by means of semireflective or dichroic mirrors to the focusing lenses of the microscope objective. The objective then focuses light within the tissue specimen or cornea at the focal plane of the objective. For in vivo confocal microscopes, the light that is scattered at the focal plane is then collected by the objective lens, passed through the semireflective mirror, and focused at a second pinhole, where it is detected. In the case of laser scanning fluorescent confocal microscopes, the light at the focal plane excites a fluorophore that emits light at a specific wavelength. This light is then collected by the objective and passed through a dichroic mirror and barrier filters that specifically transmit the desired wavelength of light. The fluorescent light is then focused on the detection pinhole to provide an image of a very small volume of the tissue. Since light passes through the entire volume of the sample, it can either scatter light or excite fluorophores throughout this illuminated volume. The light that comes from these various defocused planes (Figure 1B) when collected by the objective lens is not brought back into focus at the detection pinhole and therefore does not substantially contribute to the final image. To provide a larger field of view, several mechanisms have been employed to scan the tissue and generate a full12
sized optical slice (500 x 500 pixels). Mirrors move the light in laser scanning and slit scanning confocal microscopes, whereas multiple pinholes placed in a rotating disk are used in tandem scanning confocal microscopes. Multiple optical sections through the tissue can be obtained by moving the objective with respect to the tissue, providing observations throughout the entire tissue volume. For laser scanning fluorescent confocal microscopes, the objective or microscope stage can be moved in precise increments, and serial optical sections can be obtained to generate a stack of images that can be three-dimensionally reconstructed. For in vivo confocal microscopes that use an applanating objective lens (objectives that contact the tissue) with internal lens movements, a similar stack of images can be obtained to provide three-dimensional images. With nonapplanating objectives, rapid movement of the objective can generate a pseudo-three-dimensional data set, but the axial distance between optical sections is not precisely known. Spatial and detailed information can be obtained from whole mouse cornea with use of in vivo and laser scanning fluorescent confocal microscopes (Figure 2). In vivo confocal microscopy provides information about the light scattering structures within the tissue (Figure 2A and B). The three-dimensional reconstruction of a series of images taken through the cornea clearly shows the surface of
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the corneal epithelium and the posterior endothelial border. A change in the light scattering at the epithelialstromal interface (Figure 2A, arrow) can also be detected. Using these markers, precise measurement of the corneal, stromal and epithelial thickness can be obtained. Such measurements are useful in assessing the effects of contact lens wear on the corneal epithelium in human patients,21,22 as well as the effects of refractive surgery, including excimer laser photorefractive keratectomy14,23 and laser assisted in situ keratomileusis (LASIK).24-30 C. Interpretation of Images Figure 3. Energy transfer functions (upper) and fluorescence volume (lower) for excitation It should be noted that details under single photon (A) and multiphoton (B) conditions. Note that multiphoton fluoresconcerning the cellular structures are cence is limited to the focal plane that under optimal conditions is a 1 μm cubic volume. sometimes difficult to interpret, particularly between the surface epithelium and posterior corneal endothelium. In optical secthat can be used to study living cells using laser scanning tions taken through the mouse corneal stroma, very little confocal microscopy, such as calcein AM and 5-chlorodetail is detected (Figure 2B). Although different in vivo methylfluorescein diacetate; these initially are cell permeant confocal microscopes have varying abilities to collect scatbut nonfluorescent and then, after passing through intact tered light depending on the size of the illumination pinplasma membranes, they are cleaved by intracellular eshole or slit, increased light detection comes at the expense terases to yield highly fluorescent probes. Such probes have of reduced axial resolution. Furthermore, identifying the been used to evaluate stromal keratocyte network strucorigin of the light scattering structures is problematic and ture32,33 and the viability of corneal epithelial cells and keratocytes,34 but, to our knowledge, they have not been requires substantial investigation in order to separate celused in temporal three-dimensional studies. lular from extracellular sources. Even with use of membrane permeable fluorescent By comparison, laser scanning fluorescent confocal probes, sequential laser scanning fluorescent confocal mimicroscopy of the mouse cornea (Figure 2C and 2D) croscopic evaluation of living cells in intact tissue is probstained en bloc provides highly resolved images of struclematic. As shown in Figure 1, both in vivo and laser contures that are specifically tagged by fluorescent probes (red focal microscopy illuminate the entire volume of tissue, = intracellular actin filaments and cyan = cell nuclei). Analyresulting in excitation of fluorophores throughout the volsis of these images by fluorescence ratio imaging generume. This results in photobleaching of the fluorophore ates quantitative information on cellular structural orgaover time, making it difficult to obtain consistent data from nization; in vivo confocal microscopy can provide only a the specimen over time. Additionally, excitation light is general assessment of the light scattering properties of the phototoxic to cells, particularly at higher laser power levtissue volume. Additionally, laser scanning fluorescence els required to achieve maximal depth penetration. These microscopes can be used to measure cell density quantitalimitations markedly reduce the usefulness of using vistively by counting of nuclei throughout the tissue volume ible lasers for obtaining fluorescent images of live cells. As after adjusting for tissue swelling or thinning as shown by discussed in the next section, the development of mulPetroll et al.31 Such quantitative measurements using in vivo confocal microscopy have been attempted, but are tiphoton fluorescence confocal microscopy has eliminated not routinely used.11 or markedly reduced the effects of photobleaching and Unfortunately, conventional approaches to fluorescent phototoxicity. staining of cells and subcellular structures require fixation IV. MULTIPHOTON FLUORESCENCE and processing of tissue that not only precludes the possiCONFOCAL MICROSCOPY bility of live cell imaging but also introduces artifacts, alA. Principles and Techniques tering cell shape and structural organization. Even when Multiphoton confocal microscopy represents a major tissues are stained en bloc, tissue shrinkage or swelling readvance over visible light confocal microscopy by using mains a problem that requires the adjustment of the tissue longer wavelength infrared light to excite fluorophores that volume based on measurements of tissue thickness taken are normally excited by more phototoxic, shorter wavebefore fixation. Several vital fluorescent probes are available
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length visible light. In conventional fluorescence imaging, the absorption of light by a fluorophore leads to an elevation in the energy state of the fluorophore and a boosting of valence electrons up into a higher energy orbit from their original lower energy state (Figure 3A). After a short interval of time (about 10–8 seconds), the excited valence electrons return to the ground state and, in the process, emit a photon of light that is generally of lower energy and longer wavelength than the originally absorbed light. Each fluorophore has a characteristic excitation and emission spectra (Table 1). As practically demonstrated by Denk et al,35 multiphoton excitation results from the near simultaneous absorption of two or more photons of lower energy light that together impart sufficient energy to elevate the energy level of a single fluorophore to the excited state (Figure 3B). Multiphoton conditions are achieved when the exciting light is packed into a small enough space or high enough density to increase the probability that one molecule of fluorophore will absorb two or more photons of light within the time required for excitation (10–15 seconds). For practical considerations, the likelihood of such events occurring is markedly increased by the tight focusing of light using a laser scanning confocal microscope and by the temporal concentration of light achieved by using a femtosecond pulsed laser. In the experiments reported by Denk et al, the laser source was a self-mode locked Ti:sapphire laser that was pumped by an argon-ion laser. The wavelength of the light required to excite a fluorophore under multiphoton conditions is generally twice the wavelength required for conventional excitation though the excitation spectra for multiphoton conditions is broader than that required for single photon excitation. B. Advantages of Multiphoton Excitation
The phenomenon of two-photon or multiphoton excitation provides several breakthrough advantages over conventional excitation by either a mercury arc lamp or a visible light laser. First, lower energy, longer wavelength light can be used to excite fluorophores that ordinarily require excitation by lower wavelength, higher energy light that is more phototoxic. The use of this longer wavelength light Table 1. Excitation and Emission Characteristics for Commonly Used Fluorescent Probes Fluorophore
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Excitation Emission
Color
Hoechst 33258
360
470
Blue
Fluorescein
495
525
Green
Cy3
550
570
Red
Rhodamine
552
570
Red
Texas Red
596
620
Deep Red
Cy5
649
670
Far Red
Figure 4. Demonstration of enhanced depth of penetration that is achieved under multiphoton conditions. Images were taken in XZ from a 16-day embryo stained to identify cell nuclei in the lid (left) and lid margin (right) using visible light excitation with 488 nm laser line of the argon-krypton laser (VIS) or the 860 nm wavelength from the Verdi-Mira Ti:Sapphire laser using the descan detector or the direct, non-descan detectors.
greatly reduces the phototoxicity to the live specimens and facilitates the prolonged imaging of live cells and tissues that are required for collecting three- and four-dimensional data sets. A second advantage to using longer wavelength light is deeper penetration into thick tissue specimens due to reduced absorption of longer wavelength light, resulting in higher light intensity at deeper depths within the tissue and increased contrast and sensitivity. An example of the deeper depth penetration with improved axial resolution is provided in Figure 4. Images were obtained by scanning with a Leica SP2 multiphoton confocal microscope on a 16-day mouse embryo labeled with a nuclear stain. Images in the X-Z plane were taken using a galvanometer stage that rapidly moved the specimen in the axial plane through the surface ectoderm deeper into the developing lid tissue. As can be seen with visible laser excitation (VIS), the intensity of the signal drops substantially after passing through the first cell layer below the surface ectoderm or 25 μm into the sample. Using multiphoton excitation with the detection pin-
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hole as used for visible confocal microscopy the intensity of the signal remains higher to a depth of 3–4 cell layers or a depth of 40 μm into the tissue, a doubling of tissue depth. Without using the detection pinhole and directly detecting excited light with a non-descan detector, the overall light detection improves with no loss of signal intensity or resolution to a depth of 60 μm. A third advantage of multiphoton confocal microscopy is the improved axial resolution and reduced overall photobleaching of probes, since excitation of fluorophores occurs only at the focal plane of the objective and not throughout the illuminated volume as in single photon excitation (Figure 3A and B). Since fluorescent molecules lying above and below the focal plane are not excited, they do not contribute any signal to the detected images and therefore improve image resolution. Additionally, it is not necessary to descan the image, and the fluorescence can be detected using conventional optics or direct detection. Also, photobleaching is limited to those molecules at the focal plane. Taken together, the improved depth penetration, reduced phototoxicity, and improved axial resolution with reduced photobleaching provide dramatic improvement over conventional microscope systems for the detection and optical sectioning of cells and tissue. Of
Table 2. Excitation and Emission Characteristics of Available Fluorescent Protein Vectors Protein1
1
Excitation Emission
Color
EBFP
380
440
Blue
ECFP
433
453
Cyan
AmCyan1
458
489
Cyan
EGFP
488
509
Green
ZsGreen1
493
505
Green
EYFP
513
527
Yellow
ZsYellow1
529
539
Yellow
DsRed2
563
582
Red
HcRed1
588
618
Deep Red
Proteins are derived from the jelly fish, Aequorea victoria (EBFP, ECFP, EGFP and EYFP), or the reef coral, Anthozoa (AmCyan1, ZsGreen1, ZsYellow1, DsRed2 and HcRed1)
greater importance, however, is that multiphoton excitation provides for the possibility of three- and four-dimensional study of living cells in intact tissues. Deeper penetration and improved resolution provide the opportunity to image living cells in three dimensions when probed by appropriate fluorescent probes, particularly fluorescently linked proteins that are widely available. Reduced phototoxicity also facilitates the life-time observation of cells in three-dimensions and, thus, allows, for the first time, true four-dimensional study of cellular and tissue physiology. V. FLUORESCENT LABELING OF LIVING CELLS A. Limitations of Previously Used Probes
Figure 5. Corneal fibroblast transfected with fluorescent protein-tagged expression constructs for: A) GFP-vinculin (rabbit), B) EGFP-tubulin (Rabbit), C) EGFP-α-actinin (human), D) CFP-Moe (red) and EGFP-zyxin (green) (rabbit), and E) YFP-Fibronectin (green) deposited by a transfected cell inside a fibrillar collagen matrix (red shows reflected light image of collagen fibrils) (rabbit). The authors wish to thank Professor J Wehland and coworkers (BGF, Braunschweig, Germany) for the expression vector for EGFP-zyxin, Dr. Ken Yamada (NIH/NIDCR) for the expression vector for GFP-vinculin, Dr. Carol Otey (University of North Carolina) for the expression vector for EGFP-α-actinin, and Dr. Harold Erickson (Duke University) for the expression vectors for CFP-Moe and YFP-Fibronectin.
Many vital fluorescent, membrane permeant probes are available, but these generally require continual loading of cells and are not cell typespecific. Furthermore, these probes do not label structural intracellular and extracellular proteins. Conventional epi-fluorescence microscopy utilizes fluorescently labeled antibodies that are specific to intracellular and extracellular proteins to localize structures into and around cells. Although this approach has been an extremely valuable tool in the cell biologists armamentarium of techniques used to dissect out the cell and molecular
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human corneal fibroblasts were transfected with various vectors encoding GFP-vinculin (5A), EGFP-tubulin (5B), EGFP-α-actinin (5C) CFP-Moe and EGFP-zyxin in the same cell (5D, red and green, respectively), and YFPFibronectin (5E, green). These cells were used to study the mechanical properties of corneal fibroblasts and to measure the mechanical forces that were involved in extracellular matrix remodeling under cell culture conditions.36,39 Importantly, in vitro fourdimensional fluorescent studies using these transfected cells have shown that matrix collagen is remodeled through the exertion of mechanical forces transduced by adhesion sites between the extracellular matrix and Figure 6. Schematic showing steps involved in the transfection of corneal wound healing fibroblasts using the retroviral vector RD114. the cell plasma membrane; these adhesions are linked intracellularly to mechanisms of cell physiology and disease, most applicaactin filaments. Furthermore, cells within three-dimentions require tissue fixation and permeabilization of cells sional collagen gels can exert forces that are associated with in order for the antibodies to gain access to the intracellucontractile shortening of the actin filaments, similar to a lar or tissue structures. Additionally, antibody binding ofmuscle-like contractile mechanism. ten blocks the function of the bound molecules, renderIn these in vitro studies, cell transfection was accoming them useful in some functional studies but less promising for studying normal cell function. Therefore, in studying live cell biology, the use of antibodies is limited. B. New Labelling Techniques
Recently, several proteins have been identified from jellyfish (Aequorea Victoria) and reef coral (Anthozoa) that possess fluorescent properties. The genes for these proteins have been cloned and are now available as vectors. Several of these proteins and their excitation and emission characteristics are listed in Table 2. Using various promoters (CMV and SV40) to enhance expression, the cDNA encoding the protein can be transfected into living cells, mRNA transcribed, the proteins expressed and the cells vitally labeled. More importantly, through gene splicing, the cDNA encoding these proteins can be inserted into the cDNA encoding structural or functional proteins that, when translated, produce fluorescently tagged proteins. We have used this technique to label several proteins (W. M. Petroll, Figure 5). Briefly, cultured rabbit and 16
Figure 7. Direct fluorescent microscopy of EGFP transduced corneal wound healing fibroblasts. (A) Photomontage of fluorescent images detecting EGFP in corneal wound healing fibroblasts. Note that the cells appear in multiple clusters circumscribing the wound. (B) Higher magnification of EGFP expressing corneal fibroblasts forming a large cluster of interconnected cells. (C) A smaller cluster of EGFP expressing corneal wound healing fibroblasts. (Reprinted from Gatlin et al55 with permission of Experimental Eye Research.)
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plished by permeabilizing the cell membranes using standard transfection reagents, such as lipofectamine. Plasmid vectors carrying cDNA encoding the tagged proteins were then loaded into the cultured cells. While these approaches are useful in culture, they are less successful under in vivo conditions, where cell permeabilization may lead to substantial cell death. We have, therefore, approached this problem by using viral vector gene transfer to transduce genes into corneal cells. In recent years, significant efforts have been focused on the development of clinically applicable gene transfer systems. Several of these systems have been tested for ocular gene transfer, ranging from synthetic liposomes to recombinant viral vectors derived from adenoassociated virus, adenovirus, herpesvirus, lentiviruses and oncoretroviruses.40-50 Of particular interest have been oncoretrovirus and lentivirus vectors that have the desirable property of directing chromosomal transgene integration. This provides long-term expression in cells, and the transgene can be followed after multiple cell replications.53,54 Furthermore, both of these types of viral vectors have low ocular immunogenicity.45,46,49 Recently, we have successfully infected wound healing corneal fibroblasts in vivo, using the feline endogenous retrovirus (RD114) vector encoding the enhanced green fluorescent protein (EGFP) driven by the mouse stem cell virus (MSCV) promoter.53 Topical application of virus preparations resulted in the infection of dividing wound healing fibroblasts and the expression of EGFP by infected cells within the corneal stroma.55 The general protocol for infecting rabbit corneal wound-healing fibroblasts is illustrated in Figure 6. Different corneal wounds can be studied using this approach, including epithelial scrape injury, full thickness incisional wound and excimer laser PRK, all of which lead to infection of wound healing fibroblasts after application of vector without labeling of overlying corneal epithelium or underlying corneal endothelium. Immediately after the injury, virus suspended in serum-free media is applied to the surface of the wound and allowed to penetrate for approximately 10 minutes. During this time, viral particles penetrate into the corneal stroma and come in contact with corneal keratocytes. Over the next several days, the virus infects the keratocytes, and the encoded EGFP sequence is integrated into the keratocyte at the time of cell division. Since corneal keratocytes enter into the cell cycle sometime between 24 and 72 hours after injury, integration is probably not complete for several days. Experiments evaluating time of transfection suggest that there is no difference between infection immediately after injury and infection 24, 48 or 72 hours after injury. Once integrated, the encoded EGFP protein is then transcribed and translated, and the protein can be detected by 3 days after injury in a few cells. By 14 days, multiple clusters of positive cells can be detected at the wound margins. In lamellar keratectomy wounds, EGFP expressing cells appear to form a circumferential band at the edge of the resected lamellar button (Figure 7A, arrows). This
Figure 8. High resolution fluorescent photomicrograph of EGFP expressing corneal fibroblasts. (A and B) Wound healing fibroblast showed extensive filopodial interconnections between adjacent cells (arrows). Lamellipodia were also observed (arrowheads) that did not appear to connect to other cells. (Reprinted from Gatlin et al55 with permission of Experimental Eye Research.)
circumferential band of cells is comprised of what appears to be individual clusters of cells of various sizes from tens to hundreds of cells (Figure 7B and C). These cells appear to maintain contact with each other through extended fine dendritic processes and suggest that each cluster represents the result of clonal expansion of a single transduced cell. Higher magnification shows that cells have a diffuse fluorescence consistent with the expression of EGFP. If cells had been infected with EGFP-tagged proteins, then images would be similar to those shown in Figure 5 and would provide information about the actin organization and the adhesion structures present in the wound healing fibroblasts. Using the current construct that expresses EGFP alone, a diffuse EGFP fluorescence was detected that reveals infected corneal fibroblasts, exhibiting a broad, flattened shape with fine filipodial extensions interconnecting to adjacent cells (Figure 8A and B, arrows). Broader lamellipodia and pseudopodia can also be detected (arrowheads) that do not appear to interconnect with other cells.
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Figure 9. Modified Dikstein-Maurice chamber showing temperature heater/controller on chamber back (A, white arrow) and the teflon ring support (A, black arrowhead). Chamber back has two tubes for perfusion (B) and helps clamp the corneal scleral rim to the teflon ring so that the cornea extends out from the chamber base plate so the corneal wound is centered within the chamber (C, arrow). The chamber is then mounted on the microscope stage (D).
These results demonstrate that the RD114 pseudotyped oncoretroviral vector can infect viable rabbit corneal fibroblasts in vivo without apparent toxicity, and that transgene expression is maintained for at least 2 weeks in vivo. Furthermore, these data demonstrate that 14-day wound-healing fibroblasts are morphologically distinct from spindle-shaped, cultured fibroblasts. Using the approach of viral vector transgene expression, we have pursued the development of an ex vivo system for studying four-dimensional behavior of corneal wound healing fibroblasts.
mount (Figure 9A, black arrowhead) and the chamber back. Inserted through the optically transparent chamber back are inflow and outflow tubes (Figure 9B) for perfusing the artificial chamber that is formed. When the cornea is mounted onto the chamber, it extends out from the base of the Dikstein-Maurice chamber, as seen in Figure 9C, with the corneal wound centered within the chamber (arrow). This device is mounted onto the microscope stage of the laser scanning confocal microscope (Figure 9D). Multiphoton confocal images taken of EGFP-infected rabbit corneal wound healing fibroblasts 7 days after incisional wound injury (Figure 10) were obtained with excitation achieved using a Coherent Verdi-Mira femtosecond pulsed Ti:Sapphire laser tuned to 860 nm. Optical slices were taken every 5 μm through the anterior cornea to a depth of 75 μm from the corneal surface. The threedimensional stacks were taken every 2 minutes over a 2hour period. Three-dimensional data sets were compressed into maximum intensity projections along the XY, XZ and YZ planes. Individual planes were assembled into a single image for each time point, and the time points were assembled into a movie loop to display the temporal, threedimensional changes of the wound healing fibroblasts. Figure 10 depicts one three-dimensional, temporal plane from the data set, showing how the wound-healing fibroblasts were interconnected as they invaded into the incisional wound. Of particular interest is that the wound healing fibroblasts were organized into 3–4 cell layers, as shown in the XZ and YZ planes. Movies (not shown here) indicate that there was a slow extension of filopodia from the wound healing fibroblast that have an extension rate of 10 μm/hour. Such slow invasion was predicted by the normal time required for wound-healing fibroblasts to
VI. FOUR-DIMENSIONAL STUDY OF CORNEAL WOUND HEALING
In order to maintain normal corneal function and provide an ex vivo system that would allow continuous monitoring of corneal wound healing cells in their native environment, we have adapted the Dikstein-Maurice chamber to laser scanning fluorescence confocal microscopy( Figure 9). Using the basic design as originally described,5,6 we have modified the chamber back to be a heat-transducing, optically clear, glass backing that is surrounded by a thermocoupled heat transfer device (Figure 9A, white arrow) that both heats the back chamber and monitors the temperature level. This new chamber back is connected to a Corneal Warmer Controller (Bioptechs, Butler, PA) that constantly monitors the chamber back temperature and maintains a temperature set point +1 degree centigrade. The chamber back is then mounted on the standard Dikstein-Maurice chamber, which clamps the scleral rim surrounding the excised cornea between a Teflon ring 18
Figure 10. Single time point taken from a four-dimensional data set showing the maximum intensity projections in the XY, XZ and YZ plane for wound healing fibroblast expressing EGFP, 7 days after incisional injury.
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completely invade the wound, typically requiring 7 days to repopulate an incisional wound in the rabbit.56 Thus far, our studies suggest that wound-healing fibroblasts in incisional wounds appear to invade as threedimensional interconnected syncytiums of cells, and not as isolated wound-healing fibroblasts. Invasion is also slow, at least under the conditions thus far investigated. Overall, these findings challenge the conventional view that wound healing involves migration of isolated fibroblasts throughout the wound, which, in turn, reorganize matrix and contract wounds. Although additional experiments are needed to more fully understand the four-dimensional biology of wound healing, the combined use of multiphoton confocal fluorescence microscopy coupled with gene transfer technology may provide a clear view of this complex and important process. VII. FUTURE STUDIES The approach of vitally labeling living cells using gene transfer has great potential for studying live cell biology in intact tissues. As shown in this paper, this paradigm can be easily applied to wound healing studies using retroviral vectors to follow the function and cell fate of wound-healing fibroblasts and other cell types. Using alternative vectors, including the adeno-associated and lentiviral vectors, approaches to infection can be developed to study normal cell physiology and response to disease. Alternatively, transgenic approaches, as discussed in an earlier review by Kao et al,57 can be used to label cells for developmental studies. Furthermore, these fluorescent proteins can be driven by tissue-specific promoters to get expression into specific cell types, including corneal epithelium using the K12 promoter and corneal keratocytes using the keratocan promoter. Currently, transgenic mice are available that constitutively express EGFP and will be useful to study development and wound healing.58 REFERENCES 1. Maurice DM. Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 1968;24:1094-5 2. Maurice DM. A scanning slit optical microscope. Invest Ophthalmol Vis Sci 1974;13:1033-7 3. Koester CJ. Scanning mirror microscope with optical sectiong characteristics: applications to ophthalmology. Appl Optics 1980;19:1749-57 4. Gallagher B,Maurice DM. Striations of light scattering in the corneal stroma. J Ultrastructure Res 1977;61:100-14 5. Dikstein S, Maurice DM. The active control of corneal hydration. Isr J Med Sci 1972;8:1523-8 6. Dikstein S, Maurice DM. The metabolic basis to the fluid pump in the cornea. J Physiol 1972;221:29-41 7. Maurice DM, Singh T. An improved method for restraining rabbits for examination of the eye. Invest Ophthalmol Vis Sci 1984;25:1220-1 8. Zhao J, Nagasaki T, Maurice DM. Role of tears in keratocyte loss after epithelial removal in mouse cornea. Invest Ophthalmol Vis Sci 2001;42:1743-9
9. Egger MD, Petran M. New reflected light microscope for viewing unstained brain and ganglion cells. Science 1967;157:305-7 10. Petran M, Hadravsky M, Egger MD, Galambos R. Tandemscanning reflected-light microscopy. J Opt Soc Am 1968;58:661-4 11. Patel SV, McLaren JW, Camp JJ, et al. Automated quantification of keratocyte density by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci 1999;40:320-6 12. Patel S, McLaren J, Hodge D, Bourne W. Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci 2001;42:333-9 13. Li HF, Petroll WM, Moller-Pedersen T, et al. Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF). Curr Eye Res 1997;16:214-21 14. Moller-Pedersen T, Vogel M, Li HF, et al. Quantification of stromal thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy. Ophthalmology 1997;104:360-8 15. Petroll WM, Cavanagh HD, Jester JV. Clinical confocal microscopy. Curr Opin Ophthalmol 1998;9:59-65 16. Bohnke M, Masters BR. Confocal microscopy of the cornea. Prog Retinal Eye Res 1999;18:553-628 17. Cavanagh HD, El-Agha MS, Petroll WM, Jester JV. Specular microscopy, confocal microscopy, and ultrasound biomicroscopy: Diagnostic tools of the past quarter century. Cornea 2000;19:712-22 18. Minsky W. Memoir on inventing the confocal scanning microscope. J Scanning 1988;10:28-38 19. Wilson T, Sheppard CJ. Theory and practice of scanning optical microscopy. 1984, London, Academic Press. 20. Sheppard CJ, Cogswell CJ. Optimization of the confocal microscopy system. Trans R Microsc Soc 1990;1:231-4 21. Imayasu M, Petroll WM, Jester JV, et al. The relation between contact lens oxygen transmissibility and binding of Pseudomonas aeruginosa to the cornea after overnight wear. Ophthalmology 1994;101:371-88 22. Ladage PM, Yamamoto K, Ren DH, et al. Effects of rigid and soft contact lens daily wear on corneal epithelium, tear lactate dehydrogenase, and bacterial binding to exfoliated epithelial cells. Ophthalmology 2001;108:1279-88 23. Moller-Pedersen T, Li HF, Petroll WM, et al. Confocal microscopic characterization of wound repair after photorefractive keratectomy. Invest Ophthalmol Vis Sci 1998;39:487-501 24. Linna TU, Vesaluoma MH, Perez-Santonja JJ, et al. Effect of myopic LASIK on corneal sensitivity and morphology of subbasal nerves. Invest Ophthalmol Vis Sci 2000;41:393-7 25. Linna TU, Vesaluoma MH, Petroll WM, et al. Confocal microscopy of a patient with irregular astigmatism after LASIK reoperations and relaxation incisions. Cornea 2000;19:163-9 26. Vesaluoma M, Perez-Santonja J, Petroll WM, et al. Corneal stromal changes induced by myopic LASIK.[erratum appears in Invest Ophthalmol Vis Sci 2000 Jul;41(8):2027]. Invest Ophthalmol Vis Sci 2000;41:369-76 27. Erie JC, Patel SV, McLaren JW, et al. Effect of myopic laser in situ keratomileusis on epithelial and stromal thickness: a confocal microscopy study. Ophthalmology 2002;109:1447-52
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FOUR-DIMENSIONAL IMAGING / Jester, et al 28. Gokmen F, Jester JV, Petroll WM, et al. In vivo confocal microscopy through-focusing to measure corneal flap thickness after laser in situ keratomileusis. J Cataract Refract Surg 2002;28:962-70 29. Lee BH, McLaren JW, Erie JC, et al. Reinnervation in the cornea after LASIK. Invest Ophthalmol Vis Sci 2002;43:3660-4 30. Mitooka K, Ramirez M, Maguire LJ, et al. Keratocyte density of central human cornea after laser in situ keratomileusis. Am J Ophthalmol 2002;33:307-14 31. Petroll WM, Boettcher K, Barry P, et al. Quantitative assessment of anteroposterior keratocyte density in the normal rabbit cornea. Cornea 1995;14:3-9 32. Poole CA, Brookes NH, Clover GM. Keratocyte networks visualised in the living cornea using vital dyes. J Cell Sci 1993;106:685-91 33. Poole CA, Brookes NH, Clover GM. Confocal imaging of the keratocyte network in porcine cornea using the fixable vital dye 5-chloromethylfluorescein diacetate. Curr Eye Res 1996;15:165-74 34. Jester JV, Li HF, Petroll WM, et al. Area and depth of surfactant-induced corneal injury correlates with cell death. Invest Ophthalmol Vis Sci 1998;39:922-36 35. Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science 1990;248:73-6 36. Petroll WM, Cavanagh HD, Jester JV. Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts. Scanning (In Press). 37. Petroll WM, Ma L. Direct, dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices. Cell Motil Cytoskeleton 2003;55:254-64 38. Petroll WM, Ma L, Jester JV. Direct correlation of collagen matrix deformation with focal adhesion dynamics in living corneal fibroblasts. J Cell Sci 2003;116:1481-91 39. Vishwanath M, Ma L, Jester, JV, et al. Modulation of corneal fibroblast contractility within fibrillar collagen matrices. Invest Ophthalmol Vis Sci 2003;44:4724-35 40. Masuda I, Matsuo T, Yasuda T, Matsuo N. Gene transfer with liposomes to the intraocular tissues by different routes of administration. Invest Ophthalmol Vis Sci 1996;37:1914-20 41. Matsuo T, Masuda I, Yasuda T, Matsuo N. Gene transfer to the retina of rat by liposome eye drops. Biochem Biophys Res Commun 1996;219:947-50 42. Flannery JG, Zolotukhin S, Vaquero MI, et al. Efficient photoreceptor-targeted gene expression in vivo by recombinant adenoassociated virus. Proc Natl Acad Sci U S A 1997;94:6916-21 43. Reichel MB, Ali RR, Hunt DM, Bhattacharya SS. Gene therapy for retinal degeneration. Ophthalmic Res 1997;29:261-8 44. Cayouette M, Behn D, Sendtner M, et al. Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases
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45.
46.
47.
48.
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52.
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55.
56.
57.
58.
responsiveness of rod photoreceptors in the retinal degeneration slow mouse. J Neurosci 1998;18:9282-93 Lewin AS, Drenser KA, Hauswirth WW, et al. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med 1998;4:967-71 Seitz B, Moreira L, Baktanian E, et al. Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo. Am J Ophthalmol 1998;126:630-9 Bennett J, Maguire AM, Cideciyan AV, et al. Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina. Proc Natl Acad Sci U S A 1999;96:9920-5 Liu X, Brandt CR, Gabelt BT, et al. Herpes simplex virus mediated gene transfer to primate ocular tissues. Exp Eye Res 1999;69:385-95 Kampmeier J, Behrens A, Wang Y, et al. Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs. Hum Gene Ther 2000;11:1-8 Lau D, McGee,LH, Zhou S, et al. Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2. Invest Ophthalmol Vis Sci 2000;41:3622-33 Green ES, Rendahl KG, Zhou S, et al. Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of FGF-5 and FGF-18. Mol Ther 2001;3:507-15 Loewen N, Fautsch MP, Peretz M, et al. Genetic modification of human trabecular meshwork with lentiviral vectors. Hum Gene Ther 2001;12:2109-19 Gatlin J, Melkus MW, Padgett A, et al. Engraftment of NOD/ SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. J Virol 2001;75:9995-9 Gatlin J, Padgett A, Melkus MW, et al. Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector. Human Gene Therapy 2001;12:1079-89 Gatlin J, Melkus MW, Padgett A, et al. In vivo fluorescent labeling of corneal wound healing fibroblasts. Exp Eye Res 2003;76:361-71 Jester JV, Petroll WM, Barry PA, Cavanagh HD. Expression of alpha-smooth muscle (alpha-SM) actin during corneal stromal wound healing. Invest Ophthalmol Vis Sci 1995;36:809-19 Kao WW-Y, Liu C-Y. The use of transgenic and knock-out mice in the investigation of ocular surface cell biology. Ocular Surf 2003;1:5-19 Okabe M. Green mice as a source of ubiquitous green cells. FEBS Lett 1997;407:313-9
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Four-Dimensional Multiphoton Confocal Microscopy: The New Frontier in Cellular Imaging JAMES V. JESTER, PHD,1 BRANT R. WARD,2 AKIRA TAKASHIMA,PHD,2 JOEL GATLIN,PHD,3 J. VICTOR GARCIA,PHD,3 H. DWIGHT CAVANAGH, MD, PHD,1 AND W. MATTHEW PETROLL, PHD1 ABSTRACT This paper reviews new developments in microscopy that combine gene transfer technology, multiphoton confocal fluorescence microscopy, live cell imaging and digital imaging techniques that provide unique insights into the complex physiological processes involved in tissue function at the cellular and subcellular level. The evolution of this novel, new technology is discussed with particular attention to earlier achievements in noninvasive ocular surface imaging. The practical basis of confocal microscopy, multiphoton confocal fluorescence microscopy, and the vital fluorescent labeling of cells in living tissues are also discussed. Additionally, one application using retroviral gene transfer to express enhanced green fluorescent protein in living wound healing fibroblasts is presented as an example of how living biology can be studied in situ in four dimensions (x, y, z, time). KEYWORDS cornea, confocal microscopy, gene transfer, keratocyte
Accepted for publication December 2003. From the Departments of Ophthalmology,1 Dematology,2 and Internal Medicine3 at the University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. Supported in part by NIH grants NEI EY07348 and EY13215 and an unrestricted grant and Senior Scientist Award (JVJ) from Research to Prevent Blindness, Inc, New York. Presented in part at the Sunday Symposium, Ocular Wound Healing: A Symposium Dedicated to Dr. David Maurice, at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 4, 2003. The authors have no proprietary interest in any product or concept discussed in this article. Single copy reprint requests to James V. Jester, PhD (address below). Abbreviations are printed in boldface where they first appear with their definitions. Corresponding author: James V. Jester, PhD, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9057. Phone: (214) 648-7215. Fax: (214) 648-8447. Email: [email protected] ©2004 Ethis Communications, Inc. All rights reserved.
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I. INTRODUCTION t is generally recognized that normal tissue physiology and disease mechanisms involve complex and dynamic sets of interactions between cells, growth factors, cytokines, and an extracellular matrix environment that regulate downstream intracellular events controlling gene expression and cell function within tissues. While conventional cell and molecular approaches using tissue culture and biochemical sampling have dramatically increased our understanding of these events, this knowledge has come from ex vivo studies which separate, isolate and otherwise dissociate cells and tissues from their native environment. This reductionist approach has led to conflicting results when cellular and molecular responses from intact tissues are compared to those obtained from tissue culture models. In an attempt to return to the biology of the tissue and organism, recent tissue culture models have employed mixed cell cultures and three-dimensional extracellular matrix constructs to study the effects of cell-cell and cellmatrix interactions in controlling cell function in a more dynamic manner. For the most part, however, the totality of these interactions in the organism is best duplicated in the intact, living tissue, necessitating the development of better noninvasive approaches to studying cell function in living systems. Recent technological breakthroughs in imaging and gene transfer provide the opportunity to study the fourdimensional biology of tissues at the cellular and molecular level without the need to remove cells from their native environment. The application of this technology to the study of ocular surface physiology and disease is based on past achievements in the development of noninvasive ocular imaging combined with developments in multiphoton confocal fluorescence microscopy and viral vector technology. The purpose of this review is to familiarize the reader with the development of past and new technologies and to demonstrate how, through the combined application, we can begin to study the four-dimensional cell physiology in intact tissue systems. Specifically, this paper will first discuss past achievements of David Maurice, whose tireless interest in the biology of the eye moved him to
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FOUR-DIMENSIONAL IMAGING / Jester, et al OUTLINE I. Introduction II. Early developments in noninvasive ocular imaging III. Confocal microscopy A. History B. Principles and techniques C. Interpretation of images IV. Multiphoton fluorescence confocal microscopy A. Principles and techniques B. Advantages of multiphoton excitation V. Fluorescent labeling of living cells A. Limitations of current probes B. New labeling techniques VI. Four-demensional study of corneal wound healing VII. Future studies
develop new methods to observe ocular biology in situ, establishing him as an early pioneer in the development of noninvasive ocular imaging. Secondly, we will discuss the general area of confocal microscopy, followed by more recent developments in multiphoton confocal fluorescence microscopy that provide high resolution images of cells in living tissues. These, when combined with appropriate fluorescent probes, can be used to generate sequential, three- and four-dimensional images of cells and subcellular structures to study cell function. We will then present one approach that we have used to label cells in the live animal with a fluorescent marker to study four-dimensional cell biology in situ. Finally, we will briefly discuss how this type of approach can be used to direct future studies and answer important questions concerning corneal cell biology and development.
neal endothelium, further instrument refinement by Maurice2 and later by Koester3 produced a scanning slit microscope design that provided a wider field of view that promised images of not only the corneal endothelium but also the epithelium and stromal keratocytes. Many of these initial images obtained with the scanning slit microscope4 have been duplicated by recently developed in vivo confocal microscopes that use different designs that essentially replicate and extend Maurice’s original achievements using a thin scanning slit. Maurice was also the first to develop a functional artificial anterior chamber, and he established the conditions required for the maintenance of normal corneal endothelial function under ex vivo conditions.5,6 Combining the specular microscope with the Dikstein-Maurice chamber provided new insights into endothelial function and corneal transparency and represented the first studies of ocular surface tissue physiology at a cellular level. New methods in animal restraint devised by Maurice to facilitate continued sequential observation of the cornea7 and the recent application of vital staining of corneal cells using fluorescent dyes have led to the development of more dynamic observations of corneal cell biology and insights into the role of tears in the induction of keratocyte cell death.8 III. CONFOCAL MICROSCOPY A. History
Maurice’s early work set the stage for a dramatic expansion in our abilities to microscopically study the ocular surface in situ. Newer approaches based on the scanning slit originally designed by Maurice and the scanning pinhole as first designed by Petran et al using confocal
II. EARLY DEVELOPMENTS IN NONINVASIVE OCULAR IMAGING
David Maurice (1922–2003) was a pioneer in the design of noninvasive instrumentation to study the ocular surface and was the first to develop the “specular microscope” and to study the corneal endothelium in the living eye.1 Using a narrow slit beam for illumination and a divided objective aperture to separate the light reflected at the corneal surface from the weaker “specular” reflection of the corneal endothelium, he generated the first noninvasive optical microscopic sections through the thickness of the whole in vivo cornea. While the initial design of this microscope provided a narrow view of the cor-
Figure 1. Diagram of confocal microscope optics that collects light from the focal plane (A) and excludes scattered or excited light from the defocused plane (B).
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optics9,10 now provide high resolution images of the ocular surface and quantitative measurements of cell density,11,12 tissue thickness,13 and light scattering.14 For a more complete review of the development and design of the confocal microscope, the reader is referred to several excellent reviews that cover in more detail the technology and instrumentation that is currently available.15-17 Briefly, the confocal microscope was invented in 1957 by Minsky, who designed an optical microscope that focused illuminating light using lenses within a small volume of the specimen and then collected light from this same volume using similar lens to focus the light back to a detector.18 Since both the illumination side and the detection side of the microscope were in the same focus, the microscope was referred to as confocal. The theoretical applications of this approach have been formally developed and extended by Wilson and Sheppard19 and Sheppard and Cogswell.20 B. Principles and Techniques
Figure 2. Representative micrographs obtained from the mouse cornea using in vivo confocal microscopy (A and B) compared to laser scanning fluorescence confocal microscopy (C and D). Both represent three-dimensional data sets shown as an orthogonal cabinet projections (A) or as an XZ projection (C). Images shown in B and D represent single 2-dimensional slices taken from the corneal stroma. Arrow in A, indicates the interface between the corneal epithelium and stroma in the mouse. Fluorescent images are from rhodamine conjugated phalloidin (Red) and Syto 50 (Cyan) to repectively stain actin filaments and cell nuclei.
As demonstrated in Figure 1, current confocal microscopes essentially use a variable-sized illumination pinhole (Figure 1A). Light from the pinhole is directed by means of semireflective or dichroic mirrors to the focusing lenses of the microscope objective. The objective then focuses light within the tissue specimen or cornea at the focal plane of the objective. For in vivo confocal microscopes, the light that is scattered at the focal plane is then collected by the objective lens, passed through the semireflective mirror, and focused at a second pinhole, where it is detected. In the case of laser scanning fluorescent confocal microscopes, the light at the focal plane excites a fluorophore that emits light at a specific wavelength. This light is then collected by the objective and passed through a dichroic mirror and barrier filters that specifically transmit the desired wavelength of light. The fluorescent light is then focused on the detection pinhole to provide an image of a very small volume of the tissue. Since light passes through the entire volume of the sample, it can either scatter light or excite fluorophores throughout this illuminated volume. The light that comes from these various defocused planes (Figure 1B) when collected by the objective lens is not brought back into focus at the detection pinhole and therefore does not substantially contribute to the final image. To provide a larger field of view, several mechanisms have been employed to scan the tissue and generate a full12
sized optical slice (500 x 500 pixels). Mirrors move the light in laser scanning and slit scanning confocal microscopes, whereas multiple pinholes placed in a rotating disk are used in tandem scanning confocal microscopes. Multiple optical sections through the tissue can be obtained by moving the objective with respect to the tissue, providing observations throughout the entire tissue volume. For laser scanning fluorescent confocal microscopes, the objective or microscope stage can be moved in precise increments, and serial optical sections can be obtained to generate a stack of images that can be three-dimensionally reconstructed. For in vivo confocal microscopes that use an applanating objective lens (objectives that contact the tissue) with internal lens movements, a similar stack of images can be obtained to provide three-dimensional images. With nonapplanating objectives, rapid movement of the objective can generate a pseudo-three-dimensional data set, but the axial distance between optical sections is not precisely known. Spatial and detailed information can be obtained from whole mouse cornea with use of in vivo and laser scanning fluorescent confocal microscopes (Figure 2). In vivo confocal microscopy provides information about the light scattering structures within the tissue (Figure 2A and B). The three-dimensional reconstruction of a series of images taken through the cornea clearly shows the surface of
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the corneal epithelium and the posterior endothelial border. A change in the light scattering at the epithelialstromal interface (Figure 2A, arrow) can also be detected. Using these markers, precise measurement of the corneal, stromal and epithelial thickness can be obtained. Such measurements are useful in assessing the effects of contact lens wear on the corneal epithelium in human patients,21,22 as well as the effects of refractive surgery, including excimer laser photorefractive keratectomy14,23 and laser assisted in situ keratomileusis (LASIK).24-30 C. Interpretation of Images Figure 3. Energy transfer functions (upper) and fluorescence volume (lower) for excitation It should be noted that details under single photon (A) and multiphoton (B) conditions. Note that multiphoton fluoresconcerning the cellular structures are cence is limited to the focal plane that under optimal conditions is a 1 μm cubic volume. sometimes difficult to interpret, particularly between the surface epithelium and posterior corneal endothelium. In optical secthat can be used to study living cells using laser scanning tions taken through the mouse corneal stroma, very little confocal microscopy, such as calcein AM and 5-chlorodetail is detected (Figure 2B). Although different in vivo methylfluorescein diacetate; these initially are cell permeant confocal microscopes have varying abilities to collect scatbut nonfluorescent and then, after passing through intact tered light depending on the size of the illumination pinplasma membranes, they are cleaved by intracellular eshole or slit, increased light detection comes at the expense terases to yield highly fluorescent probes. Such probes have of reduced axial resolution. Furthermore, identifying the been used to evaluate stromal keratocyte network strucorigin of the light scattering structures is problematic and ture32,33 and the viability of corneal epithelial cells and keratocytes,34 but, to our knowledge, they have not been requires substantial investigation in order to separate celused in temporal three-dimensional studies. lular from extracellular sources. Even with use of membrane permeable fluorescent By comparison, laser scanning fluorescent confocal probes, sequential laser scanning fluorescent confocal mimicroscopy of the mouse cornea (Figure 2C and 2D) croscopic evaluation of living cells in intact tissue is probstained en bloc provides highly resolved images of struclematic. As shown in Figure 1, both in vivo and laser contures that are specifically tagged by fluorescent probes (red focal microscopy illuminate the entire volume of tissue, = intracellular actin filaments and cyan = cell nuclei). Analyresulting in excitation of fluorophores throughout the volsis of these images by fluorescence ratio imaging generume. This results in photobleaching of the fluorophore ates quantitative information on cellular structural orgaover time, making it difficult to obtain consistent data from nization; in vivo confocal microscopy can provide only a the specimen over time. Additionally, excitation light is general assessment of the light scattering properties of the phototoxic to cells, particularly at higher laser power levtissue volume. Additionally, laser scanning fluorescence els required to achieve maximal depth penetration. These microscopes can be used to measure cell density quantitalimitations markedly reduce the usefulness of using vistively by counting of nuclei throughout the tissue volume ible lasers for obtaining fluorescent images of live cells. As after adjusting for tissue swelling or thinning as shown by discussed in the next section, the development of mulPetroll et al.31 Such quantitative measurements using in vivo confocal microscopy have been attempted, but are tiphoton fluorescence confocal microscopy has eliminated not routinely used.11 or markedly reduced the effects of photobleaching and Unfortunately, conventional approaches to fluorescent phototoxicity. staining of cells and subcellular structures require fixation IV. MULTIPHOTON FLUORESCENCE and processing of tissue that not only precludes the possiCONFOCAL MICROSCOPY bility of live cell imaging but also introduces artifacts, alA. Principles and Techniques tering cell shape and structural organization. Even when Multiphoton confocal microscopy represents a major tissues are stained en bloc, tissue shrinkage or swelling readvance over visible light confocal microscopy by using mains a problem that requires the adjustment of the tissue longer wavelength infrared light to excite fluorophores that volume based on measurements of tissue thickness taken are normally excited by more phototoxic, shorter wavebefore fixation. Several vital fluorescent probes are available
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length visible light. In conventional fluorescence imaging, the absorption of light by a fluorophore leads to an elevation in the energy state of the fluorophore and a boosting of valence electrons up into a higher energy orbit from their original lower energy state (Figure 3A). After a short interval of time (about 10–8 seconds), the excited valence electrons return to the ground state and, in the process, emit a photon of light that is generally of lower energy and longer wavelength than the originally absorbed light. Each fluorophore has a characteristic excitation and emission spectra (Table 1). As practically demonstrated by Denk et al,35 multiphoton excitation results from the near simultaneous absorption of two or more photons of lower energy light that together impart sufficient energy to elevate the energy level of a single fluorophore to the excited state (Figure 3B). Multiphoton conditions are achieved when the exciting light is packed into a small enough space or high enough density to increase the probability that one molecule of fluorophore will absorb two or more photons of light within the time required for excitation (10–15 seconds). For practical considerations, the likelihood of such events occurring is markedly increased by the tight focusing of light using a laser scanning confocal microscope and by the temporal concentration of light achieved by using a femtosecond pulsed laser. In the experiments reported by Denk et al, the laser source was a self-mode locked Ti:sapphire laser that was pumped by an argon-ion laser. The wavelength of the light required to excite a fluorophore under multiphoton conditions is generally twice the wavelength required for conventional excitation though the excitation spectra for multiphoton conditions is broader than that required for single photon excitation. B. Advantages of Multiphoton Excitation
The phenomenon of two-photon or multiphoton excitation provides several breakthrough advantages over conventional excitation by either a mercury arc lamp or a visible light laser. First, lower energy, longer wavelength light can be used to excite fluorophores that ordinarily require excitation by lower wavelength, higher energy light that is more phototoxic. The use of this longer wavelength light Table 1. Excitation and Emission Characteristics for Commonly Used Fluorescent Probes Fluorophore
14
Excitation Emission
Color
Hoechst 33258
360
470
Blue
Fluorescein
495
525
Green
Cy3
550
570
Red
Rhodamine
552
570
Red
Texas Red
596
620
Deep Red
Cy5
649
670
Far Red
Figure 4. Demonstration of enhanced depth of penetration that is achieved under multiphoton conditions. Images were taken in XZ from a 16-day embryo stained to identify cell nuclei in the lid (left) and lid margin (right) using visible light excitation with 488 nm laser line of the argon-krypton laser (VIS) or the 860 nm wavelength from the Verdi-Mira Ti:Sapphire laser using the descan detector or the direct, non-descan detectors.
greatly reduces the phototoxicity to the live specimens and facilitates the prolonged imaging of live cells and tissues that are required for collecting three- and four-dimensional data sets. A second advantage to using longer wavelength light is deeper penetration into thick tissue specimens due to reduced absorption of longer wavelength light, resulting in higher light intensity at deeper depths within the tissue and increased contrast and sensitivity. An example of the deeper depth penetration with improved axial resolution is provided in Figure 4. Images were obtained by scanning with a Leica SP2 multiphoton confocal microscope on a 16-day mouse embryo labeled with a nuclear stain. Images in the X-Z plane were taken using a galvanometer stage that rapidly moved the specimen in the axial plane through the surface ectoderm deeper into the developing lid tissue. As can be seen with visible laser excitation (VIS), the intensity of the signal drops substantially after passing through the first cell layer below the surface ectoderm or 25 μm into the sample. Using multiphoton excitation with the detection pin-
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hole as used for visible confocal microscopy the intensity of the signal remains higher to a depth of 3–4 cell layers or a depth of 40 μm into the tissue, a doubling of tissue depth. Without using the detection pinhole and directly detecting excited light with a non-descan detector, the overall light detection improves with no loss of signal intensity or resolution to a depth of 60 μm. A third advantage of multiphoton confocal microscopy is the improved axial resolution and reduced overall photobleaching of probes, since excitation of fluorophores occurs only at the focal plane of the objective and not throughout the illuminated volume as in single photon excitation (Figure 3A and B). Since fluorescent molecules lying above and below the focal plane are not excited, they do not contribute any signal to the detected images and therefore improve image resolution. Additionally, it is not necessary to descan the image, and the fluorescence can be detected using conventional optics or direct detection. Also, photobleaching is limited to those molecules at the focal plane. Taken together, the improved depth penetration, reduced phototoxicity, and improved axial resolution with reduced photobleaching provide dramatic improvement over conventional microscope systems for the detection and optical sectioning of cells and tissue. Of
Table 2. Excitation and Emission Characteristics of Available Fluorescent Protein Vectors Protein1
1
Excitation Emission
Color
EBFP
380
440
Blue
ECFP
433
453
Cyan
AmCyan1
458
489
Cyan
EGFP
488
509
Green
ZsGreen1
493
505
Green
EYFP
513
527
Yellow
ZsYellow1
529
539
Yellow
DsRed2
563
582
Red
HcRed1
588
618
Deep Red
Proteins are derived from the jelly fish, Aequorea victoria (EBFP, ECFP, EGFP and EYFP), or the reef coral, Anthozoa (AmCyan1, ZsGreen1, ZsYellow1, DsRed2 and HcRed1)
greater importance, however, is that multiphoton excitation provides for the possibility of three- and four-dimensional study of living cells in intact tissues. Deeper penetration and improved resolution provide the opportunity to image living cells in three dimensions when probed by appropriate fluorescent probes, particularly fluorescently linked proteins that are widely available. Reduced phototoxicity also facilitates the life-time observation of cells in three-dimensions and, thus, allows, for the first time, true four-dimensional study of cellular and tissue physiology. V. FLUORESCENT LABELING OF LIVING CELLS A. Limitations of Previously Used Probes
Figure 5. Corneal fibroblast transfected with fluorescent protein-tagged expression constructs for: A) GFP-vinculin (rabbit), B) EGFP-tubulin (Rabbit), C) EGFP-α-actinin (human), D) CFP-Moe (red) and EGFP-zyxin (green) (rabbit), and E) YFP-Fibronectin (green) deposited by a transfected cell inside a fibrillar collagen matrix (red shows reflected light image of collagen fibrils) (rabbit). The authors wish to thank Professor J Wehland and coworkers (BGF, Braunschweig, Germany) for the expression vector for EGFP-zyxin, Dr. Ken Yamada (NIH/NIDCR) for the expression vector for GFP-vinculin, Dr. Carol Otey (University of North Carolina) for the expression vector for EGFP-α-actinin, and Dr. Harold Erickson (Duke University) for the expression vectors for CFP-Moe and YFP-Fibronectin.
Many vital fluorescent, membrane permeant probes are available, but these generally require continual loading of cells and are not cell typespecific. Furthermore, these probes do not label structural intracellular and extracellular proteins. Conventional epi-fluorescence microscopy utilizes fluorescently labeled antibodies that are specific to intracellular and extracellular proteins to localize structures into and around cells. Although this approach has been an extremely valuable tool in the cell biologists armamentarium of techniques used to dissect out the cell and molecular
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human corneal fibroblasts were transfected with various vectors encoding GFP-vinculin (5A), EGFP-tubulin (5B), EGFP-α-actinin (5C) CFP-Moe and EGFP-zyxin in the same cell (5D, red and green, respectively), and YFPFibronectin (5E, green). These cells were used to study the mechanical properties of corneal fibroblasts and to measure the mechanical forces that were involved in extracellular matrix remodeling under cell culture conditions.36,39 Importantly, in vitro fourdimensional fluorescent studies using these transfected cells have shown that matrix collagen is remodeled through the exertion of mechanical forces transduced by adhesion sites between the extracellular matrix and Figure 6. Schematic showing steps involved in the transfection of corneal wound healing fibroblasts using the retroviral vector RD114. the cell plasma membrane; these adhesions are linked intracellularly to mechanisms of cell physiology and disease, most applicaactin filaments. Furthermore, cells within three-dimentions require tissue fixation and permeabilization of cells sional collagen gels can exert forces that are associated with in order for the antibodies to gain access to the intracellucontractile shortening of the actin filaments, similar to a lar or tissue structures. Additionally, antibody binding ofmuscle-like contractile mechanism. ten blocks the function of the bound molecules, renderIn these in vitro studies, cell transfection was accoming them useful in some functional studies but less promising for studying normal cell function. Therefore, in studying live cell biology, the use of antibodies is limited. B. New Labelling Techniques
Recently, several proteins have been identified from jellyfish (Aequorea Victoria) and reef coral (Anthozoa) that possess fluorescent properties. The genes for these proteins have been cloned and are now available as vectors. Several of these proteins and their excitation and emission characteristics are listed in Table 2. Using various promoters (CMV and SV40) to enhance expression, the cDNA encoding the protein can be transfected into living cells, mRNA transcribed, the proteins expressed and the cells vitally labeled. More importantly, through gene splicing, the cDNA encoding these proteins can be inserted into the cDNA encoding structural or functional proteins that, when translated, produce fluorescently tagged proteins. We have used this technique to label several proteins (W. M. Petroll, Figure 5). Briefly, cultured rabbit and 16
Figure 7. Direct fluorescent microscopy of EGFP transduced corneal wound healing fibroblasts. (A) Photomontage of fluorescent images detecting EGFP in corneal wound healing fibroblasts. Note that the cells appear in multiple clusters circumscribing the wound. (B) Higher magnification of EGFP expressing corneal fibroblasts forming a large cluster of interconnected cells. (C) A smaller cluster of EGFP expressing corneal wound healing fibroblasts. (Reprinted from Gatlin et al55 with permission of Experimental Eye Research.)
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plished by permeabilizing the cell membranes using standard transfection reagents, such as lipofectamine. Plasmid vectors carrying cDNA encoding the tagged proteins were then loaded into the cultured cells. While these approaches are useful in culture, they are less successful under in vivo conditions, where cell permeabilization may lead to substantial cell death. We have, therefore, approached this problem by using viral vector gene transfer to transduce genes into corneal cells. In recent years, significant efforts have been focused on the development of clinically applicable gene transfer systems. Several of these systems have been tested for ocular gene transfer, ranging from synthetic liposomes to recombinant viral vectors derived from adenoassociated virus, adenovirus, herpesvirus, lentiviruses and oncoretroviruses.40-50 Of particular interest have been oncoretrovirus and lentivirus vectors that have the desirable property of directing chromosomal transgene integration. This provides long-term expression in cells, and the transgene can be followed after multiple cell replications.53,54 Furthermore, both of these types of viral vectors have low ocular immunogenicity.45,46,49 Recently, we have successfully infected wound healing corneal fibroblasts in vivo, using the feline endogenous retrovirus (RD114) vector encoding the enhanced green fluorescent protein (EGFP) driven by the mouse stem cell virus (MSCV) promoter.53 Topical application of virus preparations resulted in the infection of dividing wound healing fibroblasts and the expression of EGFP by infected cells within the corneal stroma.55 The general protocol for infecting rabbit corneal wound-healing fibroblasts is illustrated in Figure 6. Different corneal wounds can be studied using this approach, including epithelial scrape injury, full thickness incisional wound and excimer laser PRK, all of which lead to infection of wound healing fibroblasts after application of vector without labeling of overlying corneal epithelium or underlying corneal endothelium. Immediately after the injury, virus suspended in serum-free media is applied to the surface of the wound and allowed to penetrate for approximately 10 minutes. During this time, viral particles penetrate into the corneal stroma and come in contact with corneal keratocytes. Over the next several days, the virus infects the keratocytes, and the encoded EGFP sequence is integrated into the keratocyte at the time of cell division. Since corneal keratocytes enter into the cell cycle sometime between 24 and 72 hours after injury, integration is probably not complete for several days. Experiments evaluating time of transfection suggest that there is no difference between infection immediately after injury and infection 24, 48 or 72 hours after injury. Once integrated, the encoded EGFP protein is then transcribed and translated, and the protein can be detected by 3 days after injury in a few cells. By 14 days, multiple clusters of positive cells can be detected at the wound margins. In lamellar keratectomy wounds, EGFP expressing cells appear to form a circumferential band at the edge of the resected lamellar button (Figure 7A, arrows). This
Figure 8. High resolution fluorescent photomicrograph of EGFP expressing corneal fibroblasts. (A and B) Wound healing fibroblast showed extensive filopodial interconnections between adjacent cells (arrows). Lamellipodia were also observed (arrowheads) that did not appear to connect to other cells. (Reprinted from Gatlin et al55 with permission of Experimental Eye Research.)
circumferential band of cells is comprised of what appears to be individual clusters of cells of various sizes from tens to hundreds of cells (Figure 7B and C). These cells appear to maintain contact with each other through extended fine dendritic processes and suggest that each cluster represents the result of clonal expansion of a single transduced cell. Higher magnification shows that cells have a diffuse fluorescence consistent with the expression of EGFP. If cells had been infected with EGFP-tagged proteins, then images would be similar to those shown in Figure 5 and would provide information about the actin organization and the adhesion structures present in the wound healing fibroblasts. Using the current construct that expresses EGFP alone, a diffuse EGFP fluorescence was detected that reveals infected corneal fibroblasts, exhibiting a broad, flattened shape with fine filipodial extensions interconnecting to adjacent cells (Figure 8A and B, arrows). Broader lamellipodia and pseudopodia can also be detected (arrowheads) that do not appear to interconnect with other cells.
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Figure 9. Modified Dikstein-Maurice chamber showing temperature heater/controller on chamber back (A, white arrow) and the teflon ring support (A, black arrowhead). Chamber back has two tubes for perfusion (B) and helps clamp the corneal scleral rim to the teflon ring so that the cornea extends out from the chamber base plate so the corneal wound is centered within the chamber (C, arrow). The chamber is then mounted on the microscope stage (D).
These results demonstrate that the RD114 pseudotyped oncoretroviral vector can infect viable rabbit corneal fibroblasts in vivo without apparent toxicity, and that transgene expression is maintained for at least 2 weeks in vivo. Furthermore, these data demonstrate that 14-day wound-healing fibroblasts are morphologically distinct from spindle-shaped, cultured fibroblasts. Using the approach of viral vector transgene expression, we have pursued the development of an ex vivo system for studying four-dimensional behavior of corneal wound healing fibroblasts.
mount (Figure 9A, black arrowhead) and the chamber back. Inserted through the optically transparent chamber back are inflow and outflow tubes (Figure 9B) for perfusing the artificial chamber that is formed. When the cornea is mounted onto the chamber, it extends out from the base of the Dikstein-Maurice chamber, as seen in Figure 9C, with the corneal wound centered within the chamber (arrow). This device is mounted onto the microscope stage of the laser scanning confocal microscope (Figure 9D). Multiphoton confocal images taken of EGFP-infected rabbit corneal wound healing fibroblasts 7 days after incisional wound injury (Figure 10) were obtained with excitation achieved using a Coherent Verdi-Mira femtosecond pulsed Ti:Sapphire laser tuned to 860 nm. Optical slices were taken every 5 μm through the anterior cornea to a depth of 75 μm from the corneal surface. The threedimensional stacks were taken every 2 minutes over a 2hour period. Three-dimensional data sets were compressed into maximum intensity projections along the XY, XZ and YZ planes. Individual planes were assembled into a single image for each time point, and the time points were assembled into a movie loop to display the temporal, threedimensional changes of the wound healing fibroblasts. Figure 10 depicts one three-dimensional, temporal plane from the data set, showing how the wound-healing fibroblasts were interconnected as they invaded into the incisional wound. Of particular interest is that the wound healing fibroblasts were organized into 3–4 cell layers, as shown in the XZ and YZ planes. Movies (not shown here) indicate that there was a slow extension of filopodia from the wound healing fibroblast that have an extension rate of 10 μm/hour. Such slow invasion was predicted by the normal time required for wound-healing fibroblasts to
VI. FOUR-DIMENSIONAL STUDY OF CORNEAL WOUND HEALING
In order to maintain normal corneal function and provide an ex vivo system that would allow continuous monitoring of corneal wound healing cells in their native environment, we have adapted the Dikstein-Maurice chamber to laser scanning fluorescence confocal microscopy( Figure 9). Using the basic design as originally described,5,6 we have modified the chamber back to be a heat-transducing, optically clear, glass backing that is surrounded by a thermocoupled heat transfer device (Figure 9A, white arrow) that both heats the back chamber and monitors the temperature level. This new chamber back is connected to a Corneal Warmer Controller (Bioptechs, Butler, PA) that constantly monitors the chamber back temperature and maintains a temperature set point +1 degree centigrade. The chamber back is then mounted on the standard Dikstein-Maurice chamber, which clamps the scleral rim surrounding the excised cornea between a Teflon ring 18
Figure 10. Single time point taken from a four-dimensional data set showing the maximum intensity projections in the XY, XZ and YZ plane for wound healing fibroblast expressing EGFP, 7 days after incisional injury.
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completely invade the wound, typically requiring 7 days to repopulate an incisional wound in the rabbit.56 Thus far, our studies suggest that wound-healing fibroblasts in incisional wounds appear to invade as threedimensional interconnected syncytiums of cells, and not as isolated wound-healing fibroblasts. Invasion is also slow, at least under the conditions thus far investigated. Overall, these findings challenge the conventional view that wound healing involves migration of isolated fibroblasts throughout the wound, which, in turn, reorganize matrix and contract wounds. Although additional experiments are needed to more fully understand the four-dimensional biology of wound healing, the combined use of multiphoton confocal fluorescence microscopy coupled with gene transfer technology may provide a clear view of this complex and important process. VII. FUTURE STUDIES The approach of vitally labeling living cells using gene transfer has great potential for studying live cell biology in intact tissues. As shown in this paper, this paradigm can be easily applied to wound healing studies using retroviral vectors to follow the function and cell fate of wound-healing fibroblasts and other cell types. Using alternative vectors, including the adeno-associated and lentiviral vectors, approaches to infection can be developed to study normal cell physiology and response to disease. Alternatively, transgenic approaches, as discussed in an earlier review by Kao et al,57 can be used to label cells for developmental studies. Furthermore, these fluorescent proteins can be driven by tissue-specific promoters to get expression into specific cell types, including corneal epithelium using the K12 promoter and corneal keratocytes using the keratocan promoter. Currently, transgenic mice are available that constitutively express EGFP and will be useful to study development and wound healing.58 REFERENCES 1. Maurice DM. Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 1968;24:1094-5 2. Maurice DM. A scanning slit optical microscope. Invest Ophthalmol Vis Sci 1974;13:1033-7 3. Koester CJ. Scanning mirror microscope with optical sectiong characteristics: applications to ophthalmology. Appl Optics 1980;19:1749-57 4. Gallagher B,Maurice DM. Striations of light scattering in the corneal stroma. J Ultrastructure Res 1977;61:100-14 5. Dikstein S, Maurice DM. The active control of corneal hydration. Isr J Med Sci 1972;8:1523-8 6. Dikstein S, Maurice DM. The metabolic basis to the fluid pump in the cornea. J Physiol 1972;221:29-41 7. Maurice DM, Singh T. An improved method for restraining rabbits for examination of the eye. Invest Ophthalmol Vis Sci 1984;25:1220-1 8. Zhao J, Nagasaki T, Maurice DM. Role of tears in keratocyte loss after epithelial removal in mouse cornea. Invest Ophthalmol Vis Sci 2001;42:1743-9
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