Probing Multiscale Collagenous Tissue by Nonlinear Microscopy

Article pubs.acs.org/journal/abseba

Probing Multiscale Collagenous Tissue by Nonlinear Microscopy Sheng-Lin Lee, Yang-Fang Chen,* and Chen-Yuan Dong* Department of Physics, National Taiwan University, Taipei 106, Taiwan ABSTRACT: Imaging of thick biological samples has been very challenging because of severe light scattering. The transparent cornea with the unique organization of stromal collagen makes it a good candidate for deep imaging and is responsible for mechanical strength and optical clarity of the eye. However, limitation on traditional histology method provides incomplete spatial information and details on the structural organization of corneal tissue is still not sound. Second harmonic generation (SHG) microscopy is a noninvansive and nonstained technique to characterize the macromolecular organization of collagen in biological tissues. Through the combination of SHG microcopy and optimized Fourier-transform analysis, adult and embryonic chick corneas are investigated. Our results show that the anterior stroma demonstrates a fanlike distribution of rotated fibrous lamellae. In comparison with the anterior structure, the posterior stroma maintains a nonrotating pattern while increasing the depth of corneal tissue. In particular, the rotational pattern in anterior stroma exhibits a potential role of corneal maturation. Moreover, SHG microscopy in combination with the Fourier-transform-based analysis exhibits a useful tool in determination of collagen alignment in biological tissues and discrimination of diseases. KEYWORDS: cornea stroma, collagen, second harmonic generation, Fourier transform

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INTRODUCTION

The stroma is the major part of the cornea, in which collagen fibrils are distributed uniformly. The stroma which accounts for 95% of the corneal thickness in humans is characterized by parallel running lamellae which form the extracellular matrix. In comparison with collagen fibril morphology and organization in tendon (parallel) and skin (randomlike), the corneal stromal collagen fibers (lamellae) are organized into a highly intertwined three-dimensional meshwork of transverse oriented fibers that increases stiffness of stroma and controls corneal shape. The alignment of lamellae are thought to be altered through different factors including surgery, intraocular pressure alterations, thermal damage, pathologies, edema, and cellular reorganization in the three-dimensional environment.25 Accordingly, the stromal architecture of corneal collagen has a strong impact on corneal functions and quantitative techniques to investigate spatial lamellar distribution of cornea play a key role in understanding structural information on cornea under different circumstances and diseases. Recent studies on transverse arrangement of collagen lamellae with depth have used nonlinear optical microscopy to show the extent of lamellar inclination angles relative to the stroma surface and to demonstrate lamellar branching as a function of corneal depth

Second-harmonic generation (SHG) is a nonlinear secondorder optical process occurring in noncentrosymmetric systems with a large hyperpolarizability. With the advance of imaging technology, SHG microscopy has emerged as a powerful tool for imaging hierarchical organization of collagen from molecular scale up to tissue architectural level. The distributions of anisotropic biological structures with large hyperpolarizability,1,2 such as collagen,2 muscle,3,4 or microtubules,5 can be detected from SHG images, and collagen-rich tissues6 such as cornea,7,8 tendon,9,10 and arteries11 have been successfully imaged through the modality. Apart from the above-mentioned, applications of SHG in pathologies including cancer,12,13 fibrosis,14,15 and connective tissue disorders16−18 demonstrate the potential for clinical diagnosis. Furthermore, efforts have been made to analyze the fibril organization of extra cellular matrix (ECM) in the SHG images.19−22 The ECM has important roles in regulating the development, wound healing, and normal organ homeostasis. In addition to providing physical support for cells, the ECM, with its remarkable structural and biochemical diversity and functional versatility, is also involved in the establishment, separation, and maintenance of differentiated tissues and organs.23,24 Collagen, the major insoluble fibrous protein in the ECM of connective tissue, is involved in the mechanical support of tissues and is the single most abundant protein in mammals. Different arrangements between the composition and organization of collagen fibrils generate distinct tissue types and characteristics. © XXXX American Chemical Society

Special Issue: Multiscale Biological Materials and Systems: Integration of Experiment, Modeling, and Theory Received: September 19, 2016 Accepted: November 22, 2016

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DOI: 10.1021/acsbiomaterials.6b00556 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering from fine intervals of the entire thickness.26−28 In addition, Xray scattering techniques had been utilized to investigate human cornea29,30 and sclera.31 However, corneal studies of crosssection provide limited spatial information on structure and tiny structures in the collagenous stroma are fully revealed by X-ray scattering. As a step toward our goal of developing techniques to diagnose disease and injury in the cornea, we tested the hypothesis that the depth-dependent structure of corneal lamella could be quantified noninvasively using SHG microscopy and optimized Fourier-transform techniques. In this study, we show the organized pattern found in the anterior stroma can be a representative index to describe the corneal maturation during embryonic development and after birth.

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peak-related angle in the entire image stack produced the relationship curve between corneal thickness and pitch angle variation.

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RESULTS Second-Harmonic-Generated Signal Detection of Collagen-Rich Tissue. Figure 1 shows the second-harmonic

METHODS

Preparation of Specimens. Experiments were performed in accordance with the approved guidelines, in agreement with the Institutional Animal Care and Use Committee (IAUCC) of National Taiwan University. Excised pieces of rat tail tendon, mouse cornea, fish cornea, adult chicken cornea, and chick embryonic cornea (CEC) were used in this study. The collagen-rich tissues from rat and mouse were prepared after death immediately. Eyes of adult chicken and fish cornea were acquired from a local market. Post-mortem of the adult chick was within 6−8 h but that of the fish was within 2 h. Embryonic eyes were also prepared and tested at the 15th, 17th, and 19th culturing days before birth. They were fixed immediately after being humanely scarified. All eyes were fixed and preserved in the 10% paraformaldehyde Adult chick corneas were trephined centrally with a 8 mm diameter punch (Harris UniCore punch; Ted Pella, Inc., Redding, CA, USA) from fixed eyes. Corneas were also marked with scissors on the temporal side to identify the specimen’s orientation. When doing experiments, all the samples were enclosed in a confinement chamber attached to a cover slide and sealed with a cover glass and high-vacuum grease. SHG Image Acquisition. SHG images of the biological specimens were acquired using a homemade multiphoton microscopic system based on a commercial inverted microscope (TE2000U, Nikon, Japan). The excitation source was a titanium-sapphire laser (Tsunami, Spectra Physics, Mountain View, CA) pumped by a diode-pumped, solid-state (DPSS) laser system (Millennia Pro, Spectra Physics). The excitation wavelength used was 780 nm. The epi-illuminated signal was collected by the focusing objective (S Flour, 20×, NA 0.75, WI, Nikon), and filtered by the appropriate band-pass filter. The collected signals were spectrally resolved by combining dichroic mirrors (405dcxr, 530dcxr Chroma Technology) and filters (HQ390/ 10, HQ540/70, HQ630/70). In the transmission geometry, the forward second harmonic generation were collected by a lens (25 mm focal length) and resolved by a dichroic mirror (535dcxr) and filter (HQ390/10). Each image 230 × 230 μm2 in area was scanned at 256 × 256 pixel resolution by using the air immersion objective. All signals were detected by single-photon-counting photomultiplier tubes (R7400P, Hamamatsu, Hamamatsu City, Japan). Fiber Orientation Analysis. After acquiring the depth-dependent SHG images, fast Fourier transform (FFT) analysis was performed on the images at each depth. In this work, Fourier Transform technique was used to detect the orientation of collagen fibers and track the variation of corneal lamellae in three dimensions. Images were processed by FFT and converted into the corresponding frequency domain image. The same procedures were repeated in the entire image stack. To get the optimized FFT images, we calculated the principal direction from the optimized images. The optimized images were processed with custom-development programs based on the MATLAB R2010a (Mathworks, Natick, MA, USA) to compute the principal direction through angular distributions. Continuous tracking of the

Figure 1. Forward and Backward SHG images of different tissues. (a− c) Forward-scattered and (d−f) backscattered SHG images of (a, d) rat tail tendon, (b, e) mouse corneal stroma taken in the anterior stroma, and (c, f) fish corneal stroma also taken in the anterior stroma (bar = 50 μm).

generation (SHG) microscopy of forward-scattered and backward-scattered taken from rat tail tendon, mouse cornea, and fish cornea. SHG from the tendon is extremely bright with high-quality and good contrast characteristics in both backward and forward SHG. SHG-scattered images from the tendon clearly display parallel characteristics of collagen fibrils. However, images of the backscattered signals from the anterior stroma of both mouse and fish do not fully resolve individual collagenous fibers in comparison with the forward-scattered images. Although the mouse cornea shows short lamellar bands arrange in groups of collagen fibers running in random orientations, collagenous orthogonal pattern are detected in the fish cornea. Depth-Dependent Organization of Corneal Tissue. To investigate the spatial organization of corneal stroma, we took images containing the full thickness of cornea from adult chicks. Figure 2 demonstrates stromal information from the adult chicken. The organization of chicken cornea shows the similar orthogonal pattern found in the fish cornea. But the space between collagen seems to be tighter. When taking images of the entire corneal thickness, signals from three different channels, including autofluorescence, forward SHG, and backward SHG, are simultaneously recoded. The stack images with autofluorescence and forward-scattered channels are displayed in Figure 2a. The autofluorescent channel represents the distributed karatocytes in the corneal tissue. The forwardscattered SHG records the signals from the collageneous lamellae of corneal stroma. Signals of autofluorescence and forward-scattered are much brighter because they are close to the surface of the cornea and the objective. This indicates the B


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Figure 3. Directional characteristics of different tissue can be quantified. (a) Tendon with parallel collagen alignment demonstrates strong SHG signals. (b) Cornea with orthogonal pattern. Because of the FFT nature, the magnitude spectrum of both tissues display a 90° shift perpendicular to the original fiber direction. The optimized image after thresholding strengthens the directionality and represents the predominant fiber distribution.

Figure 2. Image stack of entire corneal stroma and illustrations at different depth. SHG images of corneal stroma in an adult chicken at different depth were reconstructed into stacked images to cover the entire stroma from anterior to posterior. (a) The image stack of cellular autofluorescence and forward-scattered SHG of corneal stroma. (b) The forward-scattered and backward-scattered SHG taken close to the surface and acquired in the middle of the anterior stroma (bar = 50 μm).

signals are stronger and brighter near the surface but dimmer and darker while increasing the depth into corneal stroma. Representative image of backward-SHG and forward SHG at different depth within the anterior stroma is displayed in Figure 2b. The anterior stroma close to the surface shows orthogonal interwoven arrangement of short and narrow collagen bundles. At deeper locations, an orthogonal interwoven arrangement of the collagen with larger collagen bundles is visualized. To locate the boundary of stroma and reduce possible losses of signal because of corneal thickness, we imaged target tissue blocks epithelium to endothelium and then repositioned them to scan from endothelium to epithelium. In both cases, evaluations on the size of the block and the organization of the collagen lamellae in the same block are maintained almost the same. FFT Analysis in Determining Collagen Orientation. To quantitatively determine the predominant orientation of the collagen fibril in the biological tissue, we processed the SHG images from rat tail tendon and adult chick cornea by 2D FFT analysis. The result is shown in Figure 3. The processing needs four steps including scanning the original SHG image, transforming the raw image into FFT frequency domain, thresholding the magnitude spectrum of FFT image and finding the peaks corresponding to the predominant angle of the distribution. When the tendon was placed in a horizontal direction along the long axis of the tissue, the forward-scattered SHG displayed a fairly uniform distribution of collagen fiber orientations. After processing the tendon image, one peak is found close to 90° and shown in Figure 3a. Performing the same procedures in the analysis of cornea, two peaks, close to zero or 90° are found in the magnitude spectrum as shown in the Figure 3b. Distinguishable Structural Differences Existing between Anterior and Posterior Stroma. Figures 4 and 5 show a series of corneal stromal SHG images from the adult chicken at different depths spaced approximate 50 μm. Depth-

Figure 4. Illustration of anterior stroma at different depth. Imaging of the anterior stroma starts at the surface of cornea, which is also the 0 mm reference. The thresholding image of anterior stroma exhibits constant alteration of orientation while increasing the depth.

dependent structural variation of anterior stroma is shown in the Figure 4 and posterior stroma is displayed in Figure 5. The predominant orientation of each SHG is determined by the same procedures explained in the Figure 3. Figure 4 demonstrates the orientation of anterior stroma continued alternating with the increase of corneal thickness. Hence, the magnitude spectrum of FFT image after thresholding is rotational with the increasing depth. This indicates that the alignment of corneal stroma in the anterior cornea constantly changes with the corneal thickness. However, Figure 5 C


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demonstrates the posterior stroma does not behave the same and therefore the magnitude spectrum of FFT image after thresholding exhibits a nonrotational appearance. This indicates that the stromal structure of posterior cornea maintains the same lamellar orientation. Because of the limit of current configurations, the transition between anterior and posterior stroma is found to be very thin (close to 5−15 μm). Rotational Pattern in the Anterior Stroma Indicates Corneal Maturation. To expand our understanding of current knowledge in corneal stroma, FFT-based techniques of SHG are used to quantify the anterior corneal stroma of the adult and embryonic cornea. The concept of scanning block is shown in the Figure 6a. Predominant angles detected from the optimized FFT image are sequentially recorded with the increase of thickness and displayed as a relationship curve. A typical relationship curve was plotted in Figure 6b. The 0 mm reference corresponds to the first Z-position where SHG signal becomes visible, which is always located close to the Bowman membrane. Because there are two peaks corresponding to the predominant orientation of corneal stroma, the peaks as a function of corneal thickness lead to two curves red and blue in the plot of Figure 6b. The comparison of angular displacement at different position of corneal stroma is illustrated in the Figure 6c, where 0.68°/μm is at the central cornea and 0.72°/μm is at the peripheral position. To test if the rotational pattern measured in the adult cornea exists in the embryos, we also measured the angular displacement and normalized it. The result is displayed in Figure 6d. Because corneal thickness constantly changes during the development, the comparisons here are done by measuring the corneal volume at the start position of 10 μm after the Bowman membrane toward the endothelium with the height of 100 ± 10 μm. In comparison with adult cornea, the percentage of angular displacement in the embryonic stroma was below 40% at the incubation of day

Figure 5. Illustration of posterior stroma at different depth. Same cornea as shown in the Figure 4 but with the posterior stroma presents here. The thresholding image of posterior stroma exhibits no significant rotation with the changing depth.

Figure 6. Depth-dependent relationship curve and quantification analysis. (a) Individual image from the entire image stack of corneal stroma is converted into the frequency domain to show the predominant direction of the focused plane in the stroma. Arrows of blue and red indicate the two predominant directions of collagen lamellae. (b) The predominant directions found are recorded and plotted as a function of corneal thickness. (c) The angular alterations with varying thickness at different positions of the central and peripheral cornea are compared. The central cornea is varied with 0.68 ± 0.09°/μm and the peripheral cornea is varied with 0.72 ± 0.1°/μm (N = 5). The change of peripheral cornea is slightly faster than the change of the central one. Similar protocols are done in the embryonic cornea and compared to adult chick to indicate the possible corneal maturation between the embryo and adult cornea. D


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for further measurement are definitely necessary to achieve meaningful statistics. The depth-dependent characteristics of structure are also important in medical application or treatment. Biomechanical properties of corneal stroma indicate that the anterior stroma is stiffer and behave with greater adhesion force than the posterior stroma.41The differences in the proteoglycan composition, collagen cross-linking, and keratocyte density were generally thought to relate to the weakness in the posterior stroma. These findings demonstrate regional differences existing in stromal fibril orientation and suggest that their distributions may affect corneal material behaviors and thereby the cornea’s response to intraocular pressure. Understanding these differences is important because depth-dependent structural differences and mechanical responses of the cornea are essential parameters involved in diagnosis and treatment of corneal disease, such as diagnosing and treating post-LASIK ectasia for granular corneal dystrophy, etc. Other factors considered before performing our tests are discussed in the following. We utilize forward SHG signals to identify the collagen alignment because forward corneal SHG imaging has been shown to provide better fibrillary architecture than those from the backward SHG imaging. Another reason is that the FFT-based analysis saves a lot of scanning time. The SHG characterization, namely the polarization curve, is usually done by recording the relationship of second harmonic intensity and input polarization for all polarization components of SHG. These polarization curves can be used to determine the symmetry of the sample. However, to produce accurate curve one requires more angles of polarization to get images at the same focused plane. The advantage of performing an FFT is that low signal-to-noise (SNR) data can still be used to obtain the required information. Also, the image scanning for FFT needs only once. This indicates that sufficient data can be obtained by low SNR image and fast measurements. Judging from these considerations, although optical anisotropic characteristics investigated by polarization microscopy have been valuable for the study of the oriented organization of collagen fibers in tissues, the current choice of FFT-based analysis is effective and less time-consuming. The interepithelial detachment, acuolar degenerative changes and separation of collagen fibers of corneal stroma are in general the common post-mortem changes in the cornea. Also, the corneal endothelium display swelling, lysis and detachment after death. Those observed situations exhibit the golden time to preserve the corneal tissue which is important to experimental designs and specimen preparations.

15 and day 17 but the percentage approached 60% at the incubation of day 19.

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DISCUSSION Our study shows that femtosecond pulsed infrared lasers can be used to generate second-harmonic signals from corneal collagen and therefore obtain lamellar organization of the cornea. Second-harmonic generation (SHG) microscopy, with its ability to provide submicrometer three-dimensional spatial resolution, label-free identification and characterization of images is a popular tool in biology and medicine. One of the major applications of SHG is to show the structural changes in fiber organization resulting from such diseases as keratoconus and myocardial scar.32 Keratoconus is a progressive, noninflammatory, and bilateral ectatic corneal disease. The altered biomechanical properties lead to progressive thinning and steepening of the paracentral cornea. Keratoconus corneas in general exhibit a marked decrease in lamellar interweaving and a loss of lamellae connecting to Bowman’s layer. Two-dimensional Fourier analysis has been extensively used to quantify the organization of collagen fibrils in the dermis,33,34 tendon or ligament,35,36 and cornea.37 The ligament study with the fiber direction corresponding to regions of damage displays the significant difference between normal and damaged tissue.38 In our previous research on Keratoconus, 2D FFT has been used to determine the fiber direction through measuring the aspect ratio (AR) of the elliptical shape.37 This is done by thresholding magnitude-spectrum to produce an elliptical projection, which allows for quantification by an aspect ratio (AR) of the major and minor axes of the ellipse. In this study, we demonstrate that our new approach can be effectively used to investigate the three-dimensional and depth-dependent corneal tissue structure in both the adult and embryonic cornea. Our developed method makes three-dimensional imaging possible in evaluating the collagen orientation and biomechanical strength in clinical applications.27 A recent study demonstrated that lamellar branching is found in higher vertebrates with the descending order, such that birds > reptiles > amphibians > fish. The study also illustrated that rotational patterns were found in those species.39 In fact, a gradual rotating tendency of collagen lamellae with depth in the embryonic chicken cornea has already been detected through the electron microscope.40 However, biological sample preparation for electron microscopy is complex and timeconsuming. In comparison with electron microscopy, there are no multiple preparations on sectioning and processing tissues for SHG. We attempt to fill the gap of corneal development between embryos and adult chicks. However, there are still some uncontrolled factors and drawbacks in our preliminary results shown in the Figure 6d. First, the species of embryos and the adult chick are not the same. We guarantee the embryos (white Leghorn) but not the current adults (Arbor Acres). Different species may lead to structural differences and therefore produce variation of the corneal stroma. Second, the number of embryos and adult cornea may not be enough to show the significant meaning statistically. In fact, the process of embryonic development involves a sequence of developmental events in which the embryonic cornea undergoes major structural transformations. The process ultimately determines how tissues form and function precisely. There are still many factors that regulate the entire process. We provide a chance to step forward and hopefully monitor and understand more on the natural process. Therefore, ongoing experimental designs

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CONCLUSION We present a quantitative second-harmonic generation (SHG) imaging technique based on FFT analysis that detects the 2D spatial organization of collagen fiber samples. The depthdependent lamellar arrangement of corneal stroma can be effectively measured by the modality. The lamellar organization of the avian cornea exist in two obvious regions in the stroma. Comparisons between the embryonic and adult corneas indicate a new index that corneal maturation may be represented by the rotational pattern on the anterior stroma.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail:[email protected]. Tel.: +886-2-3366-5244. E


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Sheng-Lin Lee: 0000-0001-5180-682X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this research was provided by Ministry of Science and Technology (MOST-104-2112-M-002-018-MY3), National Science Council, Taiwan (NSC104- 2112-ME-018-MY3, NSC102- 2221-E-002-030-MY3 and NSC 101-2112-M-002008-MY3) and the Center for Quantum Science and Engineering of National Taiwan University (CQSE102R891401).

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Probing Multiscale Collagenous Tissue by Nonlinear Microscopy

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