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Analytical Chem JoVE (Open Access) Citable · DOI

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

DOI: 10.3791/54648-v
What you'll learn
  • ✓ Extract and purify collagen fibers from rat tail tissue
  • ✓ Isolate elastin fibers from bovine nuchal ligament
  • ✓ Apply Brillouin spectroscopy to measure elastic properties of ECM proteins
  • ✓ Interpret micromechanical data from collagen and elastin fiber spectra
Protocol

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Difficulty
advanced
Total time
~4–6 hours per set of samples (tissue extraction, fiber preparation, spectroscopy acquisition)
Model organism
Rat (tail collagen); Bovine (nuchal ligament elastin)
Biosafety
BSL-1

Steps

1
Extract collagen fibers from rat tail

Isolate and purify type I collagen fibers from rat tail tissue using trypsin digestion to remove non-collagenous proteins. This yields high-purity collagen fiber preparations suitable for mechanical analysis.

▶ 00:46
2
Extract elastin fibers from bovine ligament

Isolate elastin fibers from bovine nuchal ligament through enzymatic and chemical extraction protocols. The resulting purified elastin fibers are prepared for comparative spectroscopic measurement.

▶ 02:16
3
Mount fibers and acquire Brillouin spectra

Mount individual collagen and elastin fibers onto reflective substrates and configure the Brillouin light scattering setup. Acquire high-resolution spectral data to measure elastic properties of each fiber type.

▶ 03:44
4
Analyze micromechanical properties from spectra

Process acquired Brillouin spectra to extract full elastic moduli and other micromechanical parameters. Compare elastic properties between collagen and elastin fibers to validate the protocol.

▶ 04:55
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