Intracellular protein conformation measured with coherent Raman microspectroscopy

Rheology experiments probe the bulk properties of a material and shear or tensile tests are based on the assumption that the sample in itself is homogeneous. As we have shown in chapter 3, this is an oversimplified picture for fibrin hydrogels and most probably for other biopolymer networks, too. A crucial aspect in measuring the sample response to a deformation is the application of stress at a well-defined location. When the spatial distribution of load in a sample matters, such as in hydrogel networks or when study- ing force distribution within the extracellular matrix, the usual shear (and tensile) geometries might not be appropriate. For microspectroscopy of fibrin (but other hydrogel samples as well) I therefore suggest testing the following experimental configuration: A thin sample is prepared by casting between glass slides as described before. However, a fiber or wire of small diameter (<100 μm) and with elasticity greater that the test ma- terial is embedded in the sample during polymerization. The fiber surface will act as a nucleation site for the biopolymer formation and thereby couple the fiber to the protein gel. One could for example use a piece of (stripped) optical fiber, a nanowire or simply hair. The loose end of the fiber can be attached to a micro stage or stepper motor while the hydrogel sample is fixed. Pulling on the string will then create a con- trolled one-dimensional shear force locally in the sample resulting in a stress distribution as shown in Fig.6.1B. Such an experiment would mimic physiological relevant scenarios like deformation of at a wound site by platelets or shear forces from blood flow (up to 38 Pa, 10000 /s). The local sample deformation can be tracked by embedded microbeads especially as the shear strain has a lateral gradient in this configuration. It would be interesting to relate the shear force to local secondary structure measured by BCARS in fibrin hydrogels of different concentration and cross-linking.