Non linear optics for the study of human scar tissue

Collagen fibers are an essential component of the dynamic process of scarring, which accompanies various diseases. Scar tissue may reveal different morphologic expressions, such as hypertrophic scars or keloids. Collagen fibers can be visualized by fluorescent light when stained with eosin. Second Harmonic Generation (SHG) creates a non linear signal that occurs only in molecules without inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices. The aim of this study was to describe the methodology for the analysis of the density and texture of collagen in keloids, hypertrophic scars and conventional scars. Samples were examined in the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas. The images were acquired in a multiphoton microscopy LSM 780-NLO Zeiss 40X. Both signals, two-photon fluorescence (TPEF) and SHG, were excited by a Mai-Tai Ti:Sapphire laser at 940 nm. We used a LP490/SP485 NDD filter for SHG, and a BP565-610 NDD filter for fluorescence In each case, ten images were acquired serially (512x512 mu m) in Z-stack and joined together to one patchwork-image. Image analysis was performed by a gliding-box-system with in-house made software. Keloids, hypertrophic scars and normal scar tissue show different collagen architecture. Inside an individual case differences of the scar process may be found between central and peripheral parts. In summary, the use of nonlinear optics is a helpful tool for the study of scars tissue. Tissue repair is a common event in the life of an organism and dysregulation of the involved systems can lead to abnormal scarring such as keloids and hypertrophic scar formation, which have different clinical as well as microscopic characteristics. Keloid is clinically defined as abnormal voluminous scar formation growing beyond the boundaries of the original wound without spontaneous regression [1, 2]. Keloids are unique to humans and occur in response to surgery, burns, or other forms of dermal injury or inflammatory conditions [3]. On the other hand, hypertrophic scars are characterized as voluminous scars confined to the margins of the original lesion which usually will show spontaneous regression several months later [4]. Abnormal scar formations have an undesirable physical and psychological impact, hampering the quality of life of the patients [5, 6]. Therefore the study of abnormal scar formation is of clinical importance. On microscopic examination abnormal scars reveal proliferation of fibroblasts and excessive deposition of collagen fibers with distortion of the architecture [7]. Collagen fibers can be visualized by fluorescent light when stained with eosin. Two-photon excitation (TPE) fluorescence microscopy is a high-resolution laser scanning imaging technique with the advantage of an improved signal-to-background ratio and the capacity of imaging at greater depths than single-photon confocal laser scanning microscopy [8]. Additional morphological information can be provided by second-harmonic generation (SHG) microscopy which has already been widely used for imaging non-centrosymmetric molecules in tissue [9-10] and cells [11-12]. When combined, the two techniques provide an important tool for the imaging of tissue sections or in vivo [8, 13-15]. SHG is particularly strong in collagen fibers and can therefore be used as an important tool for the analysis of scars [16, 17]. Recently, SHG was applied to the investigation of collagen-fiber orientation or texture changes in corneas (18), neoplasias [8, 19, 20], or healthy tissues, such as human dermis [21, 22]. Combined TPE-SHG microscopy has been applied to skin physiology and pathology, e. g. normal [22, 23], psoriasis [24], skin neoplasias [25] and cutaneous photoaging [26]. Computerized analysis of digitalized microscopic images has shown to be an objective and reproducible method for diagnostic and prognostic purposes [27-29] and is able to detect subtle morphologic changes which cannot be recognized even by a trained observer [30,31]. Texture analysis may be performed in different ways, e. g. using features of the gray-value derived co-occurrence matrix [32, 33], variables derived from the information entropy concept [34, 35], the fractal dimension [36] or from Fourier-Transformed images [34, 37-39]. The aim of this paper is to describe a method for computerized analysis of collagen texture in keloids, hypertrophic and conventional scars. Our study was approved by the local ethics committee (N 965/2007). Biopsies of scar tissue were fixed in 4% formaldehyde, dehydrated and routinely embedded in paraffin blocks. 5 mu m-thick sections were stained with hematoxylin and eosin for further analysis. Samples were examined in the National Institute of Science and Techonology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas. Images were acquired with a multiphoton microscope LSM 780-NLO Zeiss using a 40X, oil immersion objective. Two-photon excited fluorescence (TPEF) and SHG, were excited by a Mai-Tai Ti: Sapphire laser at 940nm (Spectra Physics). We