Dark-Field X-Ray Microscopy of Immunogold-Labeled Cells

Abstract

The methods of immunolabeling make visible the presence of specific antigens, proteins, genetic sequences, or functions of a cell. In this paper we present examples of imaging immunolabels in a scanning transmission x-ray microscope using the novel method of dark-field contrast. Colloidal gold, or silver-enhanced colloidal gold, is used as a label, which strongly scatters x-rays. This leads to a high-contrast dark-field image of the label and reduced radiation dose to the specimen. The x-ray images are compared with electron micrographs of the same labeled, unsectioned, whole cell. It is verified that the dark-field x-ray signal is primarily due to the label and the bright-field x-ray signal, showing absorption due to carbon, is largely unaffected by the label. The label can be well visualized even when it is embedded in or laying behind dense material, such as the cell nucleus. The resolution of the images is measured to be 60 nm, without the need for computer processing. This figure includes the x-ray microscope resolution and the accuracy of the label positioning. The technique should be particularly useful for the study of relatively thick (up to 10 μm), wet, or frozen hydrated specimens.