Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications

Abstract

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.

Keywords: fibrillar collagen; image analysis; lung; nonlinear optical microscopy; scar tissue; second harmonic generation; skin; vessels.

Figure 1

Second-harmonic generation (SHG) is a nonlinear optical process, in which two photons interacting within a nonlinear material are effectively “combined” to form a new photon with twice the energy (2$\mathsf{\omega }$), and therefore twice the frequency, or half the wavelength of the initial photons.

Figure 2

A sketch of the concept of phase matching. The fundamental wave at frequency $\mathsf{\omega }$ has a well defined phase and amplitude everywhere in the crystal. The induced dipoles all radiate at a frequency 2$\mathsf{\omega }$ with a phase dictated by the fundamental wave. The picture shows the case where all dipoles radiate in phase in the forward direction so that all contributions add up constructively.

Figure 3

Illustration of (a) collagen fibres acting as dipoles, which radiates in all directions except normal to the incident beam; and (b) the geometric arrangement of a single collagen fibre relative to an applied electric field. The emitted SHG signal after spectral filtering is shown in blue; (c) if the light is polarized along the collagen fibre axis (z), the maximum SHG signal will be observed. On the other hand, if it is polarized perpendicular to the fibre axis (x), the weakest SHG signal will be observed.

Figure 4

SHG imaging schematic and example image of mouse skin. The optical pathway schematic illustrates the general set-up for SHG imaging. The embedded mouse skin SHG image was taken using a Zeiss 710 confocal microscope was equipped with a Ti:Sa Chameleon multiphoton tunable laser (Coherent, Santa Clara, CA, USA) at 800 nm, a dichroic mirror, a custom filter set (BP:414/46, DC:495, BP:525/50), and a 20× water immersion objective. The resulting image was processed using Zen software (Zeiss Microscopy, Jena, Germany). PMT, photomultiplier tube; F-ISO, Faraday isolator.

Figure 5

Examples of label-free SHG images from different tissue and specimens. The collagen network can be observed at the dermal layer of (a) human skin; or (b) mouse skin. The collagen deposition in human airways from a (c) human healthy donor; and (d) in vitro collagen gel model, showing fibrillar collagen synthesized by human fibroblasts from airways; (e) adventitia layer of a healthy aorta artery (rabbit); and (f) scar tissue formation in infarcted hearts from mouse; (g) human cartilage; and (h) tendons from rabbits can also be assessed using SHG imaging.