Application of quantitative second-harmonic generation microscopy to posterior cruciate ligament for crimp analysis studies

Abstract

We use second-harmonic generation (SHG) microscopy to quantitatively characterize collagen fiber crimping in the posterior cruciate ligament (PCL). The obtained SHG images are utilized to define three distinct categories of crimp organization in the PCL. Using our previously published spatial-frequency analysis, we develop a simple algorithm to quantitatively distinguish the various crimp patterns. In addition, SHG microscopy reveals both the three-dimensional structural variation in some PCL crimp patterns as well as an underlying helicity in these patterns that have mainly been observed using electron microscopy. Our work highlights how SHG microscopy could potentially be used to link the fibrous structural information in the PCL to its mechanical properties.

Quantification of collagen fiber structure using second harmonic generation imaging and two‐dimensional discrete Fourier transform analysis: Application to the human optic nerve head

“…This facilitates improvements in accuracy by allowing the targeting of fibers, while excluding frequencies from other structures, in order to reduce noise . This could be exploited, for example, to characterize collagen fiber crimp . A possible future enhancement of the current method could involve the addition of semiautomated frequency filtering to attenuate undesired radial frequencies.…”

“…Notably, this approach has been found to be superior in detecting directionality of fibrillar structures when compared with other techniques such as mean intercept length and line fraction deviation methods . Several approaches have been adopted to calculate the dominant fiber orientation and anisotropy from DFT patterns, the most common being ellipse fitting to the power spectrum , and to extract the magnitude of the frequency components as an angular fiber distribution .…”

Demonstration of flat-top beam illumination in widefield multiphoton microscopy

“…For example, it is a standard practice among pathologists to rely on 2-D images obtained from thin tissue samples for pathological studies. Moreover, quantitative 2-D SHG imaging [28][29][30] and 2-D THG imaging 9 have previously been applied to assess the pathological conditions of breast tissue biopsies. In addition, 2-D imaging alone has also been shown to be useful in imaging of cultured neurons, which is typically within a few tens of micrometers.…”

“…For example, quantitative 2-D SHG and THG imaging has been used to assess the pathological conditions of breast tissue biopsies. 9,[28][29][30] In addition, 2-D imaging has also been shown to be useful in imaging of cultured neurons whose typical thickness are observed to be within a few tens of microns thereby limiting applicability of 3-D imaging. 20 Furthermore, we characterize the effect of adopting a squareshaped uniform intensity distribution on the image contrast and on quantitation of features of interest obtained using TPF and SHG imaging modalities.…”

An optomechanogram for assessment of the structural and mechanical properties of tissues

“…Furthermore, owing to the technique's specificity to structure, content in SHG images can be quantified using relatively accessible image processing methods. Indeed, this type of quantitative SHG (qSHG) imaging based on structural characterization has attracted increased interest as a reliable method for quantitative assessment of the collagen network in a variety of tissues including tendon, bone, ligament, and rat cervix 2,3,12,13 .…”