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 tissue^2,3,4. Although other ECM proteins also contribute to the stability of the fibrillar network in tendons⁵, the tendon dry mass is approximately 86% collagen

Protocol

All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) (Protocol #2013N0000062) and AAALAC guidelines at Massachusetts General Hospital. BHLHE40 null knockout or heterozygous female mice in a Scx-GFP background, age 30 days, were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Tissue preparation and fixation

1. To prepare the tissue, e.......

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.......

Materials

Name	Company	Catalog Number	Comments
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