Translator Disclaimer
1 January 2006 Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy
Author Affiliations +
A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitocondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.



Cornea is a transparent tissue covering the front surface of the eye. It protects the eye from foreign matter and delivers two thirds of the eye’s refractive power. To remain clear, cornea is devoid of any blood vessels, and is nourished by a tear film and an aqueous humor filling the chamber behind it. The arrangement, spacing, and size of collagen fibers in corneal stroma are critical in maintaining corneal transparency.1 Microbial keratitis, ocular herpes, a range of corneal dystrophies, and complications due to refractive surgeries are among many causes of corneal scars leading to permanent vision loss. Therefore, being able to visualize individual cells and collagen matrix at submicron resolution noninvasively in vivo is important in understanding the biology of normal corneas and evaluating their transformation during disease and wound healing.

In the last few years, a combination of nonlinear signals such as intrinsic two-photon fluorescence (TPF) and second harmonic generation (SHG) has been employed to form high contrast images of cells and extracellular matrix. 2, 3, 4, 5, 6, 7, 8, 9, 10 Previous nonlinear optical microscopy (NLOM) studies hinted at the possibility of employing endogenous fluorescence11 and second harmonic generation (SHG) signals to analyze corneal health,12 particularly during/after intrastromal corneal surgery.13, 14 Yet currently, no clear relationship between the observed intrinsic NLOM signals and a histologically documented corneal irregularity has been established. The sensitivity and value of these signals in elucidating cellular/extracellular matrix changes in cornea and other thick tissues resulting from aberrant gene expression are also largely unknown.

We explored the potential of NLOM to detect genetically induced phenotypic alterations in the transgenic mouse model of corneal epithelial dysfunction. The modulation of Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins, a diverse group of transcription factors] activity in epithelial tissues of mice using a keratin 14 (K14) promoter resulted in widespread epithelial abnormalities throughout the animal. The generation and characterization of these mice is discussed in a separate paper.15 The K14 dominant negative (DN)-Clim mice develop corneal abnormalities such that the corneas undergo an ultimate transformation into epidermis-like structures. These corneas become highly neovascularized, inflamed, and exhibit down-regulation of the corneal-specific protein keratin 12, concomitant with up-regulation of epidermal markers keratin 10, filaggrin, and loricrin in the epithelium.15 Eighty percent of mice are blind due to corneal opacities. In severe cases, the affected mice exhibit abnormal overgrowths of the stratified squamous epithelium, which are visually detectable and confirmed by histology.

In this work, endogenous cellular fluorescence and second harmonic signals from collagen showed dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in K14-DN-Clim mice corneas. A two-photon ratiometric redox fluorometry method based on cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) indicated about 30% higher metabolic activity in the endothelial layer compared to an epithelial layer in both normal and aberrant corneas. Overall, the K14-DN-Clim mouse is an excellent model for NLOM to explore genetically induced corneal abnormalities ranging from cellular dysplasia to stromal matrix structural irregularities and neovascularization—processes commonly observed in cancer and fibrosis.


Materials and Methods

All animal procedures were performed in accordance with an animal protocol approved by the University of California at Irvine. Mice strain CB6F1 was used in all experiments. Transgenic mice were generated by microinjection of the suitable plasmid into fertilized eggs implanted into healthy CB6F1 animals. These mice expressed the transgene in the basal cell layer of the epidermis, the outer root sheath of hair follicles, the basal cell layer of neonatal corneal epithelium, and limbal cells of adult corneal epithelium.16 RNA isolation for microarray experiments were performed as described in Ref. 17. The microarrays data were analyzed using the software Cyber-T accessible through a Web interface18 ( using a stringent statistical cut off value of 0.01.


Sample Preparation

All animals studied were euthanized by asphyxiation with CO2 . The ages of the mice were from two months up to one year. Mice eyes were removed, placed into 0.9% saline solution, and imaged immediately following euthanasia. The corneas were not dissected out of the eyes and were amenable to imaging for one or two hours. About 20 normal and 15 K14-DN-Clim mice eyes of matching ages were imaged.


Second Harmonic Generation/Two-Photon Fluorescence Imaging and Spectra

The images and spectra were obtained using a combined two-photon fluorescence (TPF)/second harmonic(SHG) setup previously described. 9, 12, 19, 20, 21, 22, 23, 24, 25, 26, 27 The excitation was linearly polarized at 766nm . Focusing objective (Zeiss, 63× water immersion, NA 1.2) was used to collect spectra and images. For photomultiplier tube (PMT) ratio imaging, a dichroic filter (500nm) and bandpass filters [ 450±25nm ; NAD(P)H and 580±30nm ; flavoprotein] were used in front of corresponding PMTs. To minimize background signal from the laser, a broadband filter (320to660nm) was placed in front of the spectrograph. The time for a typical x-y scan was one to three seconds. The spectra were collected from the single optical sections with the typical integration time of 15sec ; monochromator slit was 500μm , and the laser beam was scanned continuously during spectral acquisition. The spectra were not corrected for the instrument response. The thickness of the corneas was measured with a long working distance focusing objective (Zeiss, 40× water immersion, NA 0.8).


Histological Evaluations

After remaining in saline for approximately 3to5h during the NLOM imaging, specimens were fixed in 10% buffered formalin (Sigma-Aldrich, St. Louis, MO) overnight, dehydrated by an ethanol gradient, and embedded in paraffin wax (Fisher Scientific, Hampton, NH). The six micron sections closest to the center of the eye were stained with hematoxylin and eosin.




Second Harmonic Generation/Two-Photon Fluorescence Signals and Spectra

Figure 1 shows a hematoxylin and eosin (H and E) stained, en face histology. Epithelium (the outer region of corneal tissue, top) is a layer about five cellular layers deep and contains 98% of nerve endings, making cornea very sensitive to pain.28 Stroma (a layer behind epithelium) is the main component of cornea. Its composition is water, collagen fibers, and proteoglycans. The arrangement, spacing, and sizes of collagen fibers are critical in maintaining corneal transparency.29 Endothelium (a single layer of cells located between the stroma and the aqueous humor) mainly functions to regulate water balance. All the topographic features seen in NLOM x-y scans (Fig. 2 ) have excellent coregistration with (H and E) stained sections; however, structural organization of collagen stroma evident in SHG/TPF images is not apparent in H and E processed tissues. Regardless of animal age, the corneal size was estimated to be around 150±10μm from NLOM data, with the epithelial layer making up the first 50μm , stroma with parallel arrangement of collagen layers comprising the next 100μm (Fig. 2), and endothelial layer at 150±10μm . The endothelial layer was the last layer amenable to imaging in reflectance, and no further SHG or TPF signals were detected with a physiology long-working-distance objective (Neofluar, model 440090, Zeiss, 40× water immersion, NA 0.8). The spectrum obtained for the normal mice eyes is a broad fluorescence band (Fig. 3 ). The two peaks at around 450 and 540nm possibly result from NADH and FAD+ fluorescence, respectively (spectra not shown). The PMT ratio of the 450540-nm peaks is 30% higher for the epithelial versus endothelial layer, which we believe is due to differences in metabolic activity between the two cellular layers. The spectrum of stroma is dominated by a second harmonic signal from collagen and was described earlier.12

Fig. 1

H and E stained section of the normal mouse cornea. 1. Stratified squamous epithelium. 2. Stroma. 3. Posterior endothelium.


Fig. 2

Nonlinear optical signals (two-photon fluorescence and second harmonic generation) from a normal mouse cornea, x-y scans. The first 50μm show the spatial organization of cells within the epithelial layer. At 60μm , the parallel organization of collagen within stroma is observed. At 150μm , the endothelial layer is present.


Fig. 3

Metabolic activity in the epithelial and endothelial layers of the normal mice corneas. (a) and (b) Color images consisting of two species: NADH (blue); FAD+ (pink). (a) Epithelial layer. (b) Endothelial layer. (c) Multiphoton excited fluorescence emission spectra from epithelial and endothelial layers. The spectra are not corrected for the instrument response.


Figure 4 shows a hematoxylin and eosin (H and E) stained, en face histology section for the moderately affected K14-DN-Clim mice cornea. The high resolution NLOM images (Fig. 5 ) are highly effective in highlighting the dysplastic areas within the epithelial layer and irregular organization of collagen arrays within the stroma. In several severe cases, partial absence of the endothelial layer (differently sized cells show up in the same optical section) was also noted and confirmed by histology. When present, the endothelial cells were irregularly shaped. The spectrum for the K14-DN-Clim mice cornea is broad, with the PMT ratio of 450540-nm peaks being 30% higher for the epithelial versus endothelial layer, similar to normal corneas.

Fig. 4

H and E stained section of the K14-DN-Clim mouse cornea. 1. Epithelial cell dysplasia in the stratified squamous epithelium. 2. Cell invasion and neovascularization in stroma. 3. Posterior endothelium.


Fig. 5

Nonlinear optical signals (two-photon fluorescence and second harmonic generation) from a K14-DN-Clim mouse cornea, x-y scans. Dysplastic epithelium (center) and irregular collagen organization in the stroma (right) are clearly observed.




This work describes normal and pathological corneas in mice using reflectance NLOM (TPF and SHG combined). The epithelial layer dysplasia, irregularities in a collagen structure within stroma not apparent in H and E processed tissues, and compromised endothelial layer are reproducibly detected at high magnification in K14-DN-Clim mice corneas and validated with a conventional histology.

In many tumors, transformed epithelial cells are found to “prime” or provoke the microenvironment of extracellular matrix, which in turn undergoes dynamic alterations leading to structural changes, severe immune responses, and the formation of new blood vessels.30 During this process, the fibrillar organization of extracellular matrix proteins such as fibronectin, collagen I, III, and V, elastin, and proteoglycans is modified,31 and their overall production is increased.32 In breast, the oncofetal extracellular matrix is usually thick, flaky, and largely disorganized.33, 34 Altering the expression of the Clim gene in the epithelial tissues of mice resulted in corneas undergoing a continuous inflammatory/stromagenic/wound healing process concomitant with changes in the epithelial and endothelial cell layers. In line with up-regulation of many inflammatory markers15 and neovascularization in K14-DN-Clim mice corneas, based on mRNA microarray data (Fig. 6 ), procollagen III production is increased at least 2.5 fold compared to a healthy mouse. We therefore propose that the irregular appearance of the stromal collagen in the NLOM images of K14-DN-Clim mice corneas is due to reorganization of extracellualr matrix and deposition of smaller, disorganized fibrils perhaps associated with collagen III. While collagen I is the major fibrillar component of the cornea, it is not actively synthesized in healthy adult corneas. Collagen III is commonly overproduced in response to inflammation/wound healing in skin35 and in cornea36, 37, 38, 39 by the stromal keratocytes that transdefferentiate into fibroblasts and myofibroblasts.40 It is deposited with disorganized architecture, providing a framework for collagen I fibers41 observed in the later stages of wound repair.

Fig. 6

Procollagen, type III, alpha 1 mRNA expression is up-regulated in K14-DN-Clim mice. Data from Affymetrix microarrays for normal (WT) and transgenic (TG) mice corneas using two probe sets (P1, P2). The bars and numbers are the means; the error bars are the standard errors of the mean based on sample standard deviations.


Most K14-DN-Clim mice develop obvious macroscopic evidence of corneal abnormalities around six weeks of age. However, partial loss of Clim function leads to a complex phenotype with a severity of the condition varying greatly among individuals of the same age. To overcome the challenge of detecting early stages in K14-DN-Clim mice corneal pathology, we are currently implementing live animal imaging.

Two-photon ratiometric redox fluorometry based on cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) has been proposed as a tool to study mitocondrial energy metabolism.42 Only reduced NADH and oxidized FAD states of these cofactors are significantly fluorescent and linked through the NADH-Q-reductase and various dehydrogenases43 in a process known as oxidative phosphorylation (OXPHOS). They will respond differently to changes in mitochondrial metabolic states, and a ratio NADHFAD+ will be a measure of mitochondrial energetics. Several earlier studies used fluorescence of NAD(P)H11, 44 and flavoprotein to address the mitochondrial energy metabolism in the biological systems. 45, 46, 47, 48, 49, 50, 51, 52, 53 Our two-photon ratiometric imaging results indicate a higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from metabolites. An observed difference of 30% might be even greater in vivo, where the endothelial layer conducts metabolite exchange and actively regulates water balance at physiological temperatures. Earlier observations of flavoprotein/NADH and NAD(P)H signals obtained from single photon excitation of perfused corneas detected 50% higher metabolic rate for endothelium relative to that of epithelium.11, 44, 47 Tissue handling associated with ex-vivo measurements is likely to influence our two-photon ratiometric redox fluorometry measurements by inducing a shut down of endothelial layer function in about an hour at room temperature. Therefore, we are currently investigating the utility of functional corneal imaging on live animals.



High-resolution reflectance-based NLOM methods offer novel approaches to probe the downstream effects of gene expression and their influence on phenotypes in vivo without use of exogenous dyes. In this study, the Clim gene is partially blocked in the epithelial compartment of mice tissues: however, it causes significant structural changes in corneal stroma, indicating that Clims may be involved in epithelial-mesenchymal interactions. We detect these changes at high magnification using nondestructive NLOM imaging, and on comparison to normal stromas shows that the method is effective in detailing structural pathologies in reorganized extracellular matrix. These extracellular matrix structural alterations are likely due to collagen III synthesis and deposition during healing and remodeling of corneal stroma. Because no exogenous dyes are employed, NLOM visualization can be used in selecting tissues for gene expression profiling. In addition, NLOM ratiometric redox fluorometry results provide preliminary confirmation that cellular metabolic function can be characterized and imaged in thick tissues.


Julia G. Lyubovitsky thanks George E. Hewitt and the George E. Hewitt Foundation for Medical Research for the fellowship. This work was made possible, in part, through the NIH Laser Microbeam and Medical Program (LAMMP) at the University of California, Irvine (P41-RR01192) and by the Air Force Office of Scientific Research (AFOSR), under agreement number FA9550-04-1-0101. Bogi Andersen is supported by NIH grant AR44882.



D. M. Maurice, “The structure and transparency of the cornea,” J. Physiol. (London), 136 268 –286 (1957). 0022-3751 Google Scholar


P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J., 77 3341 –3349 (1999). 0006-3495 Google Scholar


P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt., 6 (3), 277 –286 (2001). 1083-3668 Google Scholar


L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, “Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy,” Opt. Lett., 25 320 –322 (2000). 0146-9592 Google Scholar


L. Moreaux, O. Sandre, S. Charpack, M. Blanchard-Desce, and J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J., 80 1568 –1574 (2001). 0006-3495 Google Scholar


R. Gauderon, P. B. Lukins, and S. J. Sheppard, “Optimization of second-harmonic generation microscopy,” Micron, 32 691 –700 (2001). 0968-4328 Google Scholar


P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J., 82 493 –508 (2002). 0006-3495 Google Scholar


Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, “Subsurface tumor progression investigated by noninvasive optical second harmonic tomography,” Proc. Natl. Acad. Sci. U.S.A., 96 10854 –10856 (1999). 0027-8424 Google Scholar


A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A., 99 11014 –11019 (2002). 0027-8424 Google Scholar


E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, and Y. Boucher, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med., 9 796 –801 (2003). 1078-8956 Google Scholar


D. W. Piston, B. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc., 178 20 –27 (1995). 0022-2720 Google Scholar


A. T. Yeh, N. Nassif, A. Zoumi, and B. J. Tromberg, “Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence,” Opt. Lett., 27 2082 –2084 (2002). 0146-9592 Google Scholar


M. Han, L. Zickler, G. Giese, M. Walter, F. H. Loesel, and J. F. Bille, “Second-harmonic imaging of cornea after intrastromal femtosecond laser ablation,” J. Biomed. Opt., 9 (4), 760 –766 (2004). 1083-3668 Google Scholar


M. Han, G. Giese, L. Zickler, H. Sun, and J. F. Bille, “Mini-invasive corneal surgery and imaging with femtosecond lasers,” Opt. Express, 12 4275 –4281 (2004). 1094-4087 Google Scholar


X. Xu, J. A. Spencer, K. K. Lin, E. I. Kudryavtseva, B. Andersen, “Characterization of mice expressing a dominant negative Clim in epithelial tissues,” Google Scholar


K. K. Lin, D. Chudova, G. W. Hatfield, P. Smyth, and B. Andersen, “Identification of hair cycle-associated genes from time-course gene expression profile data by using replicate variance,” Proc. Natl. Acad. Sci. U.S.A., 101 15955 –15960 (2004). 0027-8424 Google Scholar


P. Baldi and A. D. Long, “A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes,” Bioinformatics, 17 509 –519 (2001). 1367-4803 Google Scholar


A. Agarwal, M. L. Coleno, V. P. Wallace, W. Y. Wu, C. H. Sun, B. J. Tromberg, and S. C. George, “Two-photon laser scanning microscopy of epithelial cell-modulated collagen density in engineered human lung tissue,” Tissue Eng., 7 191 –202 (2001). 1076-3279 Google Scholar


A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, B. J. Tromberg, “Influence of optical properties on two photon fluorescence imaging in turbid samples,” Appl. Opt., 39 1194 –1201 (2000). 0003-6935 Google Scholar


T. F. Kelly, F. M. Sutton, V. P. Wallace, and B. J. F. Wong, “Chondrocyte repopulation of allograft cartilage: A preliminary investigation and strategy for developing cartilage matrices for reconstruction,” Otolaryngol.-Head Neck Surg., 127 256 –270 (2002). 0194-5998 Google Scholar


V. J. LaMorte, A. Zoumi, and B. J. Tromberg, “Spectroscopic approach for monitoring two-photon excited FRET from homodimers at the subcellular level,” J. Biomed. Opt., 8 (3), 357 –361 (2003). 1083-3668 Google Scholar


B. J. F. Wong, V. Wallace, M. Coleno, H. P. Benton, B. J. Tromberg, “Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage,” Tissue Eng., 7 599 –606 (2002). 1076-3279 Google Scholar


A. T. Yeh, B. Choi, J. S. Nelson, and B. J. Tromberg, “Reversible dissociation of collagen in tissues,” J. Invest. Dermatol., 121 1332 –1335 (2003). 0022-202X Google Scholar


A. T. Yeh, B. S. Kao, W. G. Jung, Z. P. Chen, J. S. Nelson, and B. J. Tromberg, “Imaging wound healing using optical coherence tomography and multiphoton microscopy in an in vitro skin-equivalent tissue model,” J. Biomed. Opt., 9 (2), 248 –253 (2004). 1083-3668 Google Scholar


A. Zoumi, “Optimization of imaging depth and spectral content in multiphoton microscopy,” 202 Univ. California, Irvine, (2003). Google Scholar


A. Zoumi, X. Lu, G. S. Kassab, and B. J. Tromberg, “Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy,” Biophys. J., 87 2778 –2786 (2003). 0006-3495 Google Scholar


D. M. Maurice, “Clinical physiology of the cornea,” Int. Ophthalmol. Clin., 2 561 –572 (1962). 0020-8167 Google Scholar


K. M. Meek, A. J. Quantock, C. Boote, C.- Y. Liu, W. W.- Y. Kao, “An x-ray scattering investigation of corneal structure in keratocan-deficient mice,” Matrix Biol., 22 467 –475 (2003). 0945-053X Google Scholar


E. Cukierman, “A visual-quantitative analysis of fibroblastic stromagnesis in breast cancer progression,” J. Mamary Gland Biol. Neoplasia, 9 311 –324 (2004). Google Scholar


T. Silzle, G. J. Randolph, M. Kreutz, and L. A. Kunz-Schughart, “The fibroblast: Sentinell cell and local immune modulator in tumor tissue,” Int. J. Cancer, 108 173 –180 (2004). 0020-7136 Google Scholar


A. Noel, F. Kebers, E. Maquoi, and J. M. Foidart, “Cell-cell and cell-matrix interactions during breast cancer progression,” Curr. Top Pathol., 93 183 –193 (1999). 0070-2188 Google Scholar


Y. C. Wong, and N. N. Tam, “Dedefferentiation of stromal smooth muscle as a factor in prostate carcinogenesis,” Dedefferentiation, 70 633 –645 (2002). Google Scholar


L. A. Kunz-Schughart and R. Knuechel, “Tumor-associated fibroblasts (Part I): Active stromal participants in tumor development and progression,” Histol. Histopathol, 17 599 –621 (2002). 0213-3911 Google Scholar


J. N. Clore, I. K. Cohen, and R. F. Diegelmann, “Quantitation of collagen types I and III during wound healing in rat skin,” Proc. Soc. Exp. Biol. Med., 161 337 –340 (1979). 0037-9727 Google Scholar


C. Cintron, B. S. Hong, H. I. Covington, and E. J. Macarak, “Heterogeneity of collagens in rabbit cornea: type III collagen,” Invest. Ophthalmol. Visual Sci., 29 767 –775 (1988). 0146-0404 Google Scholar


J. A. Anderson, P. S. Binder, M. E. Rock, and M. P. Vrabec, “Human excimer laser keratectomy. Immunohistochemical analysis of healing,” Arch. Ophthalmol. (Chicago), 114 54 –60 (1996). 0003-9950 Google Scholar


D. S. Malley, R. F. Steinert, C. A. Puliafito, and E. T. Dobi, “Immunofluorescence study of corneal wound healing after excimer laser anterior keratectomy in the monkey eye,” Arch. Ophthalmol. (Chicago), 108 1316 –1322 (1990). 0003-9950 Google Scholar


C. Chen, B. Michelini-Norris, S. Stevens, J. Rowsey, X. Ren, M. Goldstein, and G. Schultz, “Measurement of mRNAs for TGFβ and extracellular matrix proteins in corneas of rats after PRK,” Invest. Ophthalmol. Visual Sci., 41 4108 –4116 (2000). 0146-0404 Google Scholar


J. L. Funderburgh, M. M. Mann, and M. L. Funderburgh, “Keratocyte phenotype mediates proteoglycan structure (A role for fibroblasts in corneal fibrosis),” J. Biol. Chem., 278 45629 –45637 (2003). 0021-9258 Google Scholar


D. E. Birk, J. M. Fitch, J. P. Babiarz, T. F. Linsenmayer, “Collagen type I and type V are present in the same fibril in the avian corneal stroma,” J. Cell Biol., 106 999 –1008 (1998). 0021-9525 Google Scholar


S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and Flavoprotein,” Biophys. J., 82 2811 –2825 (2002). 0006-3495 Google Scholar


C. A. Combs and R. S. Balaban, “Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP),” Biophys. J., 80 2018 –2028 (2001). 0006-3495 Google Scholar


B. Masters, M. V. Riley, J. Fischbarg, and B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: Chemical analysis, non-invasive fluorometry and physiological correlates,” Exp. Eye Res., 37 1 –9 (1983). 0014-4835 Google Scholar


B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxydation-reduction states in vivo,” Science, 137 499 –508 (1962). 0036-8075 Google Scholar


B. Chance, I. A. Salkovitz, and A. G. B. Kovach, “Kinetics of mitochondrial flavoprotein and pyridine nucleotide in perfused heart,” Am. J. Phys., 223 207 –218 (1972). 0002-9505 Google Scholar


B. Chance and M. Lieberman, “Intrinsic fluorescence emission from the cornea at low temperatures: Evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides,” Exp. Eye Res., 26 111 –117 (1978). 0014-4835 Google Scholar


W. Kunz and W. Kunz, “Contribution of different enzymes to flavoprotein fluorescence in isolated rat liver mitochondria,” Biochim. Biophys. Acta, 841 237 –246 (1985). 0006-3002 Google Scholar


B. R. Masters, P. T. C. So, and E. Gratton, “Optical biopsy of in vivo human skin: Multiphoton excitation microscopy,” Lasers Med. Sci., 13 196 –203 (1998). Google Scholar


D. N. Romashko, E. Marban, and B. O’Rourke, “Sucellular metabolic transients and mitochondrial redox waves in heart cells,” Proc. Natl. Acad. Sci. U.S.A., 95 1618 –1623 (1998). 0027-8424 Google Scholar


A. V. Kuznetsov, O. Mayboroda, D. Kunz, K. Winkler, W. Schubert, and W. S. Kunz, “Functional imaging of mitochondria in saponin-permeabilized mice muscle fibers,” J. Cell Biol., 140 1091 –1099 (1998). 0021-9525 Google Scholar


A. V. Kuznetsov, Y. Usson, X. Leverve, and R. Margreiter, “Subcellular heterogeneity of mitochondrial function and dysfunction: Evidence obtained by confocal imaging,” Mol. Cell. Biochem., 256 359 –365 (2004). 0300-8177 Google Scholar


A. C. Croce, A. Ferrigno, M. Vairetti, R. Bertone, I. Freitas, and G. Bottiroli, “Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions,” Photochem. Photobiol. Sci., 3 920 –926 (2004). 1474-905X Google Scholar
© (2006) Society of Photo-Optical Instrumentation Engineers (SPIE)
Julia G. Lyubovitsky, Joel A. Spencer, Tatiana B. Krasieva, Bogi Andersen, and Bruce Jason Tromberg "Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy," Journal of Biomedical Optics 11(1), 014013 (1 January 2006).
Published: 1 January 2006

Back to Top