Cross-Correlation with Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation: Improved Imaging at the Nanoscale

Research into the extracellular matrix (EMC), a porous network structured with macromolecules and minerals which surround the cells, allows for future applications such as improved drug delivery systems. The diffraction limit of light makes structures on the scale of the wavelength of light difficult to distinguish due to the convolution of a point spread function (PSF) in widefield microscopy. Here we are using an imaging technique via microscopy called fluorescence correlation spectroscopy super-resolution optical fluctuation (fcsSOFI) to image EMC like hydrogel environments. fcsSOFI combines two previous methods (FCS and SOFI) to image both the diffusion dynamics and structure of ECM like materials. FCS utilizes auto-correlation curves to calculate diffusion coefficients. SOFI utilizes independent fluorophores and auto-correlation to gain a higher resolution with a squared point spread function. Here we are implementing cross-correlation SOFI to allow for a higher sampling rate via the creation of interpolated pixels between the original pixels (thus allowing for a four-fold increase in pixels). Since a SOFI image covers the same physical distance whether using auto or cross-correlation, second order cross-correlation doubles our sampling frequency. We can now identify structural features twice as small without any aliasing in our fcsSOFI images, allowing us to gather even more structural information about our EMC like environments at the nanoscale.