ABSTRACT
Total internal reflection fluorescence (TIRF) microscopy is a powerful means of probing biological cells because it reduces autofluorescence, but the need for direct contact between the cell surface and the microscope slide hinders chemical access to the cell surface. In this work, a submicrometer crystalline layer of colloidal silica on the microscope coverslip is shown to allow TIRF microscopy while also allowing chemical access to the cell surface. A 750 nm layer of 165 nm silica colloidal crystals was sintered onto a fused silica coverslip, and Chinese hamster ovary cells were successfully grown on this surface. This cell line over-expresses the human delta-opioid receptor, which enabled probing of the binding of a labeled ligand to the receptors on the cell surface. Total internal reflection and chemical access to the cell surface are demonstrated. The range of angles for total internal reflection is reduced only by 1/3 due to the lower index of refraction of the colloidal multilayer relative to fused silica.
Subject(s)
Colloids , Image Enhancement/methods , Microscopy, Fluorescence/methods , Silicon Dioxide , Specimen Handling/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Porosity , Surface PropertiesABSTRACT
Silica colloidal crystals require multiple processing steps before they are useful materials in analytical applications, such as chemical separations, microarrays, sensors, and total internal reflection microscopy. These chemical processing steps include calcination, sintering, surface rehydroxylation, and chemical modification, but these steps have not been fully characterized for colloidal crystals. Silica particles of nominally 200 nm in diameter were prepared, and FTIR, SEM, UV-visible spectroscopy, and refractive index measurements were used to study the changes in chemical composition, particle size, and particle density throughout the process. The final material is shown to be a durable, crack-free crystal of solid particles bearing a fully hydroxylated surface of silanols, which can then be chemically modified.