Quantification of Collagen Fiber Morphologies utilizing Multiphoton Microscopy

Morphology 1 Segment of a human abdominal aorta after optical clearing. The translucent appearance allows for an increased penetration depth during multiphoton microscopy.

Morphology 2 Collagen fiber bundles in the adventitia of a human abdominal aorta. Image taken by detecting the second-harmonic generation signal of collagen using a multiphoton microscope.

The mechanical behavior of an arterial wall is mainly governed by the organization and composition of the three major microstructural components: collagen, elastin, smooth muscle cells. Their influence on the cardiovascular function in health and disease has been the subject of extensive research. Among these components, it is collagen that endows the arterial wall with strength and load resistance, thus making it the most relevant mechanical tissue constituent.

Research indicates that changes in the mechanical properties of healthy arterial walls play a role in arterial disease and degeneration (e.g., increased stiffening of vessel walls with age, atherosclerosis, etc.). Meaningful quantifications of morphological collagen data in human arteries, therefore, are fundamental to a better understanding of the underlying mechanical principles governing the biomechanical response of vessel walls.

Collagen can be visualized by utilizing either stained histological sections or different microscopy techniques, e.g., polarized microscopy, electron microscopy, fluorescence microscopy and multiphoton microscopy (MPM), featuring enhanced penetration depth in soft biological tissues, good optical sectioning and resolution. Both, fluorescence microscopy and MPM use collagen as a source of second-harmonic generation and autofluorescence, enabling direct observation without staining.

We developed a unique method for the extraction and quantification of collagen fiber distributions from 2D images, and for a statistical analysis among varying length-scales. In aiming to move beyond a 2D quantification, a novel approach followed ultimately, which combines a new sample preparation technique for intact arterial segments with optical tissue clearing and subsequent imaging using second-harmonic generation microscopy. This approach yields 3D image stacks throughout the thickness of the arterial wall (up to 1.5 mm). The collagen structures are extracted and quantified, resulting in a representative 3D distribution of collagen fiber orientations from which structural parameters are determined in order to be utilized directly in numerical codes using fiber-reinforced material laws.

Funding: DACH Project support by Austrian Science Fund (FWF) and German Research Foundation (DFG); Graz University of Technology