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.

Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: II. Tomography

“…However, the resolution in this particular demonstration of soft X‐ray tomography is comparable to that of 3D optical microscopy, and can be expected to improve towards the 30 nm level, as demonstrated in other X‐ray microscopy experiments ( Spector et al ., 1997 ; Schneider, 1998). Labelling methods are only in their infancy in X‐ray microscopy ( Jacobsen et al ., 1993 ; Chapman et al ., 1996; Moronne, 1999), yet it is useful in many cases to be able to observe unlabelled structures in 3D as well (especially to understand the 3D relationships between different structures in a cell). Finally, 500 eV soft X‐rays are able to penetrate through 10 μm layers of ice with good contrast for small features, whereas electron microscopy of unsectioned frozen hydrated eukaryotic cells faces severe challenges because of specimen thickness.…”

Table-top water window transmission x-ray microscopy: Review of the key issues, and conceptual design of an instrument for biology

“…It has been shown that the same probes can be localized in WW-TXM. 18,19 Light emission of various materials such as P31 phosphor and fluorescent polystyrene spheres has been detected in STXM. 20 Quantum dots ͑QDs͒ are increasingly used in fluorescence microscopy.…”

Spectromicroscopy of Biological and Environmental Systems at Stony Brook: Instrumentation and Analysis

“…Imaging modalities such as differential phase contrast, 9 Nomarski differential interference contrast, 10 and dark field imaging [11][12][13][14] require the use of a spatially configured detector. (One can also record the full microdiffraction pattern from each pixel 15,16 at the cost of increased dataset size and image processing time.)…”