Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging

Abstract

The loss of organization of the corneal collagen lattice induced by photothermal effects was analyzed by using second-harmonic generation (SHG) imaging. Porcine cornea samples were treated with low-power laser irradiation in order to get localized areas of tissue disorganization. The disorder induced within the irradiated area of corneal stroma was quantified by means of Discrete Fourier Transform, auto-correlation and entropy analyses of the SHG images. Polarization modulated SHG measurements allowed to probe the changes in the structural anisotropy of sub-micron hierarchical levels of the stromal collagen. Our results emphasize the great potential of the SHG imaging to detect subtle modifications in the collagen assembly. The proposed analytical methods may be used to track several genetic, pathologic, accidental or surgical-induced disorder states of biological tissues.

Multimodal imaging to study the morphochemistry of basal cell carcinoma

“…Such intact collagen is visualized most specifically in the tissue using SHG microscopy. The capability of this method has been previously demonstrated by Cicchi et al [19,20]. In the samples investigated in this case study very different collagen structures are visible.…”

“…These structures stabilize the surrounding tissue and, hence, fulfill a structurally supporting task within the tissue. Generally, during the development of various diseases collagen structures underlie alterations which are readily detectable by means of SHG imaging [19,20]. With respect to the formation of cancer, early stage malignancies are typically surrounded by a capsule of collagen rich tissue or sits on top of the basal membrane, a natural boundary, which prevents the cancer from spreading into neighboring tissue.…”

Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy

“…The anisotropy of the image can be evaluated by measuring the ratio of the two axes of the ellipsis. In a previous work on cornea [26], the authors performed an elliptic fit on the thresholded FFT images. Here, we have used a slightly different method to measure the aspect ratio.…”

“…Collagen fibers produce a high SHG signal [18] and can be imaged inside skin dermis with SHG microscopy [4,18]. Recently, SHG was also used for investigating collagen-fiber orientation and their structural changes in fibrotic collagen [19], human dermis [20][21][22][23], keloid [24], cornea [25,26] and in the tumor microenvironment [27][28][29][30]. The combination of TPEF and SHG is particularly useful when imaging dermis tissue because the two main components of dermis (collagen and elastin) can be imaged with SHG and TPEF microscopy, respectively.…”

In vivo non‐invasive monitoring of collagen remodelling by two‐photon microscopy after micro‐ablative fractional laser resurfacing

“…A more powerful imaging modality is represented by two-photon excited fluorescence (TPF) microscopy [20], which is a high-resolution laser scanning imaging technique enabling deep optical imaging of tissues, as demonstrated by studies performed on ex vivo tissue samples [21][22][23][24][25], fresh biopsies [26][27][28][29][30][31], and also in vivo on both animals [32] and humans [33][34][35][36][37][38]. Additional morphological information can be provided by second-harmonic generation (SHG) microscopy [39][40][41][42][43][44][45][46][47][48][49], which can be combined with TPF microscopy using the same laser source. Combined TPF-SHG microscopy represents a powerful tool for imaging skin dermis, since the main dermal components, collagen and elastin, can be imaged by SHG and TPF microscopy, respectively [22].…”