Nonlabeling and quantitative assessment of chondrocyte viability in articular cartilage with intrinsic nonlinear optical signatures

Abstract

Chondrocyte viability is a crucial factor for evaluating cartilage health. Most prevalent cell viability assays rely on dyes and are not applicable for in vivo or longitudinal studies. Here we demonstrated that the two-photon excited autofluorescence and second harmonic generation microscopy provided high-resolution imaging of cartilage tissue and distinguished live/dead chondrocytes by visual assessment. Furthermore, the normalized autofluorescence ratio was proposed as a quantitative indicator to determine chondrocyte viability. Based on the indicator, a curve fitting and simulated receiver operating characteristic method was proposed to identify the live/dead cell populations as well as the indicator threshold without dye labeling. Thus, it established the label-free imaging method for chondrocyte viability assay in cartilage tissue. Impact statement Chondrocytes are the only cellular component found in the cartilage, playing a critical role in maintaining the homeostasis of articular cartilage. The viability of chondrocytes is a crucial factor for evaluating cartilage health. However, the current prevalent cell viability assays rely on dye staining and thereby are not applicable in vivo or in longitudinal assessments. In this study, we demonstrate that the intrinsic signals such as two-photon excited autofluorescence and second harmonic generation can be used to classify live and dead chondrocytes in cartilage tissue. A quantitative measure is also proposed allowing development of automated assessment algorithms. The nonlabeling nature of this method suggests the potential applicability to nondestructive and in vivo assessment of cartilage health.

Measuring chondrocyte viability of articular cartilage based on label-free two-photon microscopy and deep learning image analysis

“…The improvement is more prominent when using wMCV-Net. The contribution from the SHG channel was also found in our previous study, where populations of live and dead cells could be better separated according to the NAD(P)H and FPs ratios if the SHG channel was used to reduce the autofluorescence contribution from collagen in the ECM region 16 . The usefulness of the SHG channel in CV analysis is perhaps due to the overall improvement of the contrast between cells and the ECM area.…”

“…Our studies with freshly excised cartilage tissue from rat tibias 16 showed that live chondrocytes exhibited higher intensity of autofluorescence from NAD(P)H than dead chondrocytes did. We found that the ratio between NAD(P)H and NAD(P)H+FPs emission was a robust measure to differentiate live and dead chondrocytes.…”

“…For rat samples, entire tibias were excised from Sprague-Dawley rats (n=15) in accordance with the Institutional Animal Care and Use Committee's regulations. After removal of muscles, tibias were held by a customized clamp prepared and tibia plateaus were aligned and imaged with settings as previously described 16 . For porcine samples, hind knee joints from adult Yorkshire pigs were obtained from a local meat processing company.…”

Nanowarming and ice-free cryopreservation of large sized, intact porcine articular cartilage

“…An inverted Olympus FV1200MPE multiphoton laser scanning microscope (Olympus, Center Valley, PA) with a ×10 lens was used for imaging. The excitation wavelength was 800 nm 56 . The green fluorescence signal was collected within the 495–540 nm range while red fluorescence signal was collected in the 575–630 nm range.…”

Biomedical optical imaging technology and applications: From basic research toward clinical diagnosis

“…Li et al. 76 investigated application of intrinsic nonlinear optical imaging such as two-photon excited autofluorescence and second harmonic generation (SHG) microscopy to quantitatively assess chondrocyte viability in articular cartilage. Lu et al.…”