Single-crystal infrared (IR) spectroscopy is a promising method for protein structure analysis, where a protein crystal sample is fixed in a micro flow cell. Single-crystal calcium fluoride (CaF2) is expected as the flow cell substrate material for its excellent optical property. However, CaF2 is a highly brittle material having strong anisotropy, thus is extremely difficult to machine. Up to date, there is no available literature on fabrication of CaF2 flow cells. In this study, micro flow cells of single-crystal CaF2 were fabricated by ultraprecision cutting technology. Fly cutting was conducted using a single-crystal diamond tool having straight edges to generate a depth-varying rectangle cross section for the flow cell. The effects of cutting direction, workpiece orientation, undeformed chip thickness and tool rake angle on cutting behavior were investigated. Based on experiments and analysis, optimal conditions for ductile machining of micro grooves in CaF2 were identified. As a result, a 10 μm deep CaF2 micro flow cell with surface roughness of 2.4 nmRa was successfully fabricated. Using the fabricated flow cell, IR spectroscopic analysis of a protein single crystal at room temperature was succeeded. This study demonstrated the effectiveness of ultraprecision cutting technology in CaF2 micro flow cell fabrication, which contributes to the IR analysis of protein, and in turn, the advance of life science.Keywords : Ultraprecision cutting, Single crystal, Calcium fluoride, Ductile machining, Micro flow cell
IntroductionPhysiological processes are series of chemical reactions regulated by proteins. Protein structure analysis is important for revealing the functional mechanism of a protein at the atomic level. Conventionally, X-ray crystallography has been used for determining the atomic arrangement of a protein, whereas infrared (IR) spectroscopy is available for analyzing the chemical structure and reactivity of functional sites in the protein. Recently, Kubo et al. developed a highly-sensitive IR spectrometer, suitable for measuring protein solution (Kubo et al., 2013). On the other hand, to facilitate the complementary use of IR and X-ray crystallographic analyses, the IR measurement for protein crystals is also desirable. Previously, an IR flow cell for mounting protein crystals has been reported, where a tapered Teflon spacer was customdesigned (Sage 1997). However, because the precise taper cutting of a thin Teflon sheet is very difficult, an alternative method for the crystal sample mounting still needs to be developed. In the IR analysis of protein, conventional plastic or glass micro flow cells cannot be used due to their low transmittance of IR light. Instead, a micro flow cell made of singlecrystal calcium fluoride (CaF2) is preferential for mounting the protein sample, because CaF2 has excellent transmittance of IR light and enables precise IR measurements of proteins.Conventional plastic and glass flow cells are made by chemical etching or injection/press molding methods. However, those me...