Fluorescence microscopy sheds light on the behavior of proteins in hydrogels

Due to their biocompatibility, hydrogels are often used to study properties of proteins in biotechnical applications such as drug delivery and tissue engineering. However, because of instrumental limitations, the effects of hydrogels on proteins are not well-understood. A comprehensive tutorial by Anuj Saini and Lydia Kisley outlines fluorescence microscopy techniques for observing protein interactions with materials for bio-imaging applications. Combining two of these techniques, the tutorial presents a new method to study protein dynamics.

In fluorescence microscopy, an excited electron emits a photon as it relaxes into its ground state. During this process, fluorescently-labeled proteins can be selectively detected when embedded in hydrogels. The authors used a unique application of fluorescence microscopy that combined fluorescence correlation spectroscopy (FCS), which uses correlation analyses to study the diffusion of proteins, with super-resolution optical fluctuation imaging (SOFI), which traditionally uses blinking fluorescence to study nanoscale structures. Their combined technique, dubbed fcsSOFI, can provide nanoscale information about both the structure and diffusion of the labeled protein without some of the drawbacks of other methods.

“With a lot of current techniques, you get a static picture, or you have to freeze the sample, which distorts the material,” Kisley said. Their method works to overcome this limitation.

The researchers found a spectrum of nanopore sizes in the hydrogel as well as an order of magnitude range in protein diffusion. “What we need to get at next is the relation between the two,” said Kisley.

Looking ahead, Kisley plans to study different proteins and protein behaviors by combining other types of super-resolution techniques. “We want to head in the direction of disease-relevant proteins in biologically-relevant hydrogels instead of a model protein in a model hydrogel,” she said.

Source: “Fluorescence microscopy of biophysical protein dynamics in nanoporous hydrogels,” by Anuj Saini and Lydia Kisley, Journal of Applied Physics (2019). The article can be accessed at https://doi.org/10.1063/1.5110299 .