Quantitative second harmonic generation imaging of cartilage damage

Abstract

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples. SHG imaging of the frozen cryopreserved specimens showed a dramatic loss of mean gray value intensities when compared to both fresh and vitrified tissues (P<0.05), indicating structural changes of the extracellular matrix, in particular the deformation and destruction of the collagen fibers in the analyzed articular cartilage. A 0.9974 correlation coefficient was observed between the metabolic cell activity assessed by the alamarBlue technique, and retention of collagen structure between the three experimental groups. These studies suggest that multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling may prove to be useful tools for the investigation of extracellular matrix changes in preserved cartilage, giving insights on the structural quality prior to implantation.

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space

“…bone, tendon, cartilage, and cornea) or contain collagen as part of the extracellular matrix (ECM; e.g. skin, breast, ovary, liver, kidney, and colon) [1][2][3][4][5][6][7][8][9][10]. Similarly, SHG has been used to image the sarcomeric structure in skeletal muscle [1,11,12].…”

Measurement of Friction-induced Changes in Pig Aorta Fibre Organization by Non-invasive Imaging as a Model for Detecting the Tissue Response to Endovascular Catheters

“…The techniques of SHG and TPEF have been previously used to investigate collagen and elastin networks, respectively, in arteries [10,20] heart valves [12,21] porcine cartilage [22] and skin [23].…”

Multimodal nonlinear optical microscopy reveals critical role of kinesin-1 in cartilage development

“…Nonlinear optical (NLO) microscopy provides an ideal solution to resolve the structural characteristics in tissues and cells, featured with its inherent 3D resolution and capacity for deep penetration [19][20][21]. Recently, NLO microscopy has been used to study the chondrocytes and ECM of cartilage [22][23][24][25]. Compared with electron microscopy, a tool widely used in the study of cartilage, NLO microscopy is applicable to fresh tissues, which prevents artefacts from complicated specimen preparation.…”

“…Compared with electron microscopy, a tool widely used in the study of cartilage, NLO microscopy is applicable to fresh tissues, which prevents artefacts from complicated specimen preparation. In addition, label-free imaging based on intrinsic NLO signals in the fresh biological samples provides better preservation of biological information than using fixed or frozen specimens [22]. Therefore, it is desirable to perform NLO imaging on freshly excised samples in order to accurately interpret the biomedical characteristics of tissues.…”