Two-Photon Microscopy for the Study of Tendons
Summary
This article outlines the process of preparing, setting up, and imaging tendons using multiphoton microscopy. Additionally, it covers the application of SHG for analyzing collagen fibril alignment and the creation of a 3D representation of tendons. This methodology proves highly valuable in characterizing tendon cells and their ECM during injury and development.
Abstract
Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence.
SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.
Introduction
To properly function and transmit force from muscle to bone1, tendons rely on the intermolecular and intramolecular bonds between collagen fibers. The intricate self-assembly, crosslinking, and alignment of the collagen fibers result in the establishment of a highly organized matrix that contributes to the biomechanical strength and flexibility of tendon tissue2,3,4. Although other ECM proteins also contribute to the stability of the fibrillar network in tendons5, the tendon dry mass is approximately 86% collagen6, with collagen I making up to 96% of the total collagen content7,8. This ultimately makes collagen structure a key output of normal tendon health and function.
Some clinical imaging modalities used for tendons are MRI and/or ultrasound. While ultrasound technology provides images of the fascicular structure of the tendon and reveals some of the fibrillar structure in the tissue9, the resolution is not ideal for quantification. MRI has better spatial resolution than ultrasound imaging, but it is still limited. However, these methods are also limited in their potential imaging depth through a tissue. On a more microscale, small- and wide-angle light scattering and confocal microscopy can be used to assess the structure of collagen fibrils and any potential abnormalities.
In contrast, second-harmonic generation (SHG) microscopy differs from other imaging methods as it can capture the outer shell of the collagen fibrils. Collagen I fibrils in tendons are organized in a uniaxial parallel manner through the tendon ECM and are non-centrosymmetric in nature4,10. These properties can be leveraged for imaging using a multiphoton microscope, which can produce clear images that are up to 2-3 times deeper than confocal imaging11. This also allows us to generate better quality optical sections of the tendon. When light is projected into the sample of interest, an SHG signal is produced, and this scattering of light can be captured. In tendons, this produces an image of the collagen structure and alignment, thus allowing us to evaluate potential morphological and pathological consequences of tendinopathies, injuries, etc.
Since collagen makes up most of the tendon dry mass and contributes to tendon function6,12, disruption to collagen structure can affect the biomechanical properties of tendons. Thus, analyzing its structure can help us better understand the impact and severity of injuries, as well as create a metric for healing efficacy. This paper reviews a method that uses multiphoton and SHG microscopy to analyze how development and injuries affect the structure and alignment of collagen I fibrils and to generate 3D images of tendon ECM in animal models. Therefore, our method to image tendons may help researchers characterize tendon cells and ECM during development or after injury.
Protocol
Representative Results
Discussion
This article presents a method to prepare, dissect, and image the mouse Achilles tendon, utilizing the non-centrosymmetric crystalline properties of the tendon ECM. Key steps in tissue preparation involve permeabilization for counterstains and ensuring proper tissue placement in a petri dish during imaging. Instead of Draq5, Hoechst 33258 can be used at a 1:100,000 dilution13, but Draq5 is preferred for its high permeability, photostability, and minimal photobleaching. Proper tissue placement is c…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Jenna Galloway and the members of Galloway Lab for their support and encouragement in the development and troubleshooting of these protocols.
Materials
| 0.5 M EDTA pH 8.0 | Invitrogen | AM9262 | |
| 2 mL microcentrifuge tubes | USA Scientific | 1620-2700 | |
| 20 mL scintillation vial | Sigma-Aldrich | Z190527-1PAK | |
| 4% Paraformaldehyde | Electron Microscopy Sciences | 50-980-487 | Use PFA ampuole to create 4% PFA solution |
| 6 mm Biopsy Punch Tool | Ted Pella Inc. | 15111-60 | |
| 60 x 15 mm petri dish | |||
| BHLHE40 null knockout or heterozygous mice in a Scx-GFP background | The Jackson Laboratory | JAX ID #029732 | MGI ID #3717419 |
| Coverslips | Fisher | 12-544-F | Can use any coverslip that spans the area of the M20 washer |
| dPBS | Gibco | 14190144 | |
| Draq5 | ABCAM | ab108410 | |
| Fine scissors 21 mm cutting edge | Fine Science Tools | 14060-10 | |
| FVMPE-RS multiphoton laser scanning microscope | Olympus | ||
| Gelfoam Sterile Sponge Size 50 | Pfizer | 00009-0323-01 | |
| INSIGHT X3-OL IR pulsed laser | Olympus | ||
| MaiTai HPDS-O IR pulsed laser | Olympus | ||
| Phosphate-Buffered Saline (1x) | Invitrogen | AM9625 | Dilute 10x PBS in milli-Q water to get 1x solution |
| Stainless steel M20 flat washer | McMaster-Carr | ||
| Triton X-100 | MP Biomedicals | 807426 | Dilute Triton X-100 in dPBS to get 1% solution |
| Vannas spring scissors 4 mm cutting edge | Fine Science Tools | 15018-10 | |
| XLPlan N 25X WMP Lens |
References
- Sharma, P., Maffulli, N. Tendon injury and tendinopathy: healing and repair. The Journal of Bone and Joint Surgery. 87 (1), 187-202 (2005).
- Kadler, K. E., Holmes, D. F., Trotter, J. A., Chapman, J. A. Collagen fibril formation. The Biochemical Journal. 316 (Pt 1), 1-11 (1996).
- Butler, D. L., Grood, E. S., Noyes, F. R., Zernicke, R. F. Biomechanics of ligaments and tendons. Exercise And Sport Sciences Reviews. 6, 125-181 (1978).
- Tsai, S. L., Nödl, M. T., Galloway, J. L. Bringing tendon biology to heel: Leveraging mechanisms of tendon development, healing, and regeneration to advance therapeutic strategies. Developmental Dynamics: An Official Publication of the American Association of Anatomists. 250 (3), 393-413 (2020).
- Subramanian, A., Schilling, T. F. Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix. Development (Cambridge, England). 142 (24), 4191-4204 (2015).
- Lin, T. W., Cardenas, L., Soslowsky, L. J. Biomechanics of tendon injury and repair. Journal of Biomechanics. 37 (6), 865-877 (2004).
- Riley, G. P., Harrall, R. L., Constant, C. R., Chard, M. D., Cawston, T. E., Hazleman, B. L. Tendon degeneration and chronic shoulder pain: changes in the collagen composition of the human rotator cuff tendons in rotator cuff tendinitis. Annals of the Rheumatic Diseases. 53 (6), 359-366 (1994).
- Bobzin, L., Roberts, R. R., Chen, H. J., Crump, J. G., Merrill, A. E. Development and maintenance of tendons and ligaments. Development (Cambridge, England). 148 (8), dev186916 (2021).
- Hodgson, R. J., O’Connor, P. J., Grainger, A. J. Tendon and ligament imaging. The British Journal of Radiology. 85 (1016), 1157-1172 (2012).
- Williams, R. M., Zipfel, W. R., Webb, W. W. Interpreting second-harmonic generation images of collagen I fibrils. Biophysical Journal. 88 (2), 1377-1386 (2005).
- Centonze, V. E., White, J. G. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophysical Journal. 75 (4), 2015-2024 (1998).
- Franchi, M., Trirè, A., Quaranta, M., Orsini, E., Ottani, V. Collagen structure of tendon relates to function. The Scientific World Journal. 7, 404-420 (2007).
- Grinstein, M., Dingwall, H. L., O’Connor, L. D., Zou, K., Capellini, T. D., Galloway, J. L. A distinct transition from cell growth to physiological homeostasis in the tendon. eLife. 8, e48689 (2019).
- Rueden, C. T., Schindelin, J., Hiner, M. C., et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 18, 529 (2017).
.