Semi-automated optical tweezers technique for quantitative microrheology of the extracellular matrix.

Optical tweezers are a research technology based on the force of a precisely aligned laser beam, enabling the manipulation of cells and microparticles without mechanical contact, as well as the highly accurate measurement of interactions between microscopic particles and surrounding molecules. The extracellular matrix is a viscoelastic special structure of tissues composed of a network of charged molecules and studying it at the molecular level requires highly sensitive measurement techniques.
The aim of this doctoral research was to investigate the applicability of optical tweezers in the microrheological study of viscoelastic substances, particularly the properties of the extracellular matrix. A specific focus was on the role of heparan sulfate chains attached to basal membrane proteoglycans. The optical tweezer system was integrated with a light microscope, and a dedicated computer program was developed for analyzing the resulting image data. Since studying molecular structures at the micro- and nanometer scale is particularly sensitive to external mechanical disturbances, a computational method was also developed in this work to eliminate such interferences.
To model the extracellular matrix, the studies utilized a commercial basement membrane extract (Matrigel), and the role of heparan sulfate chains was examined by adding either extra heparan sulfate (heparin) or heparinase enzyme, which breaks down the heparan sulfate chains of proteoglycans. The microrheological properties of the basement membrane extract were measured using microbeads with different surface charges and significantly smaller, negatively charged fluorescent dextran molecules to observe their diffusion. Both the addition and the enzymatic degradation of heparan sulfate chains increased the mobility of the microbeads but reduced the microelastic properties of the basement membrane extract. The effects on dextran molecule diffusion varied depending on the treatment.
Changing the surface charge of the microbeads by adding polyethylene glycol chains weakened the interactions between the beads and the extracellular matrix and led to increased bead mobility.
The basement membrane forms a specialized extracellular matrix that acts as a support structure for cells and regulates the passage of compounds at tissue boundaries. This doctoral research provided new insights into how particle size, charge, and heparan sulfate chains affect molecular interactions in the basement membrane and the microrheological properties of the extracellular matrix. The methods developed in this study and the research conducted with them demonstrated that optical tweezer-based methods are suitable for investigating the microrheological properties of the basement membrane.