Development of Cell Staining Technique for X‐Ray Microscopy

We report a technique for detection of sub‐cellular organelles and proteins with hard x‐ray microscopy. Several metals were used for enhancing contrast for x‐ray microscopy. Osmium tetroxide provides an excellent stain for lipid and can delineate cell membrane. Uranyl acetate has high affinity for nucleotide and can stain nucleus. Immunolocalization of specific proteins and sub‐cellular organelles was achieved by 3’3 diaminobenzidine (DAB) with nickel enhancement and nanogold‐conjugated secondary antibody with silver enhancement. The x‐rays emitted from synchrotron source was monochromatized by double crystal monochromator, the photon energy was fixed at 8 keV to optimize the focusing efficiency of the zone plates. The estimated resolution is about 60 nm. When compared with visible light and conventional confocal microscopy, the X‐ray microscopy provides a superior resolution to both conventional optical microscopes.