Silica colloidal crystals as porous substrates for total internal reflection fluorescence microscopy

In cell biology and chemistry, total internal reflection microscopy (TIRFM) has proven to be a useful technique that allows the probing of cellular processes with high-signal-to-noise ratio imaging. However, samples on solid substrates limit the accessibility to probe processes on extracellular membrane surface closest to the microscope objective. Colloidal crystals provide a porous alternative to the traditional solid substrates. Thin crystals exhibit optical properties similar to that of a fused silica coverslip allowing for TIRFM in the same manner as with a typical coverslip as demonstrated by the observance of Chinese hamster ovary cells with fluorescently labeled receptors on both types of substrates. Accessibility of the cell membrane closest to the substrate and the ability to probe fluorophore orientation information was observed by the binding of TIPP-cy5 to the human delta opioid receptor.