A characterisation of dark-field imaging of colloidal gold labels in a scanning transmission X-ray microscope.

While X-ray microscopes provide images of biological specimens for which the contrast is mainly due to the difference in the absorption of carbon and oxygen when X-rays transmitted through the specimen are detected, signals other than absorption can also be used to form images. Using the Stony Brook scanning transmission X-ray microscope at the National Synchrotron Light Source, high-angle dark-field images have been formed of cells labelled with colloidal gold, with and without silver enhancement. The high density of the colloidal gold particles, or the silver particles seeded by the gold, leads to a large scattering signal, and the fact that the particle diameters are comparable to the width of the microscope point spread function results in good localisation of the label with high contrast. The dark-field images can have a greater signal to noise ratio than bright-field images acquired with the same incident X-ray dose. The theory of dark-and bright-field imaging is reviewed. Theoretical calculations of scattering from gold and silver particles are presented and good agreement is found between these and experimental dark-field images of 30 nm diameter gold particles. The signal to noise ratios of experimental bright-and dark-field images are measured and found to be in agreement with theory. Images are presented of cells labelled by immunolabelling and in situ hybridisation.