The immunogold technique is a valuable method for labeling cellular macromolecules. However, multiple labeling using colloidal gold (cAu) nanoparticles of different sizes presents certain drawbacks; namely, as particle size increases, there is a decreased labeling efficiency and diminished spatial resolution with respect to the locations of labeled epitopes. Both concerns also limit the utility of heavy metal particles for comparative analysis of labeling densities. To minimize the variables due to differential labeling efficiencies, the best solution would be to conduct multiple labeling with particles of similar size. Consequently, some parameter other than size is necessary to distinguish each label type. In this study, we report the synthesis of colloidal palladium (cPd) nanoparticles of similar size but having two distinct shapes, umbonate and faceted, which are readily distinguishable from spherical colloidal gold particles. Their utility and fidelity as labels using a human platelet whole-mount model is also demonstrated.

Physiological studies of intracellular messengers frequently employ intracellular injections of large molecules that either monitor or modulate the metabolism of the messenger cascade. Injected molecules have unknown mobility in the cytosol and unknown accessibility to various cytosolic compartments, including those postulated to be traversed by intracellular messenger molecules. In order to determine whether injected molecules have access to the confined spaces through which messenger molecules must diffuse, we injected 5-nm colloidal gold or horseradish peroxidase, or both, into Limulus ventral photoreceptors. Injections were made by applying pressure pulses to the back of an intracellular micropipette that also monitored membrane voltage. The tissue was fixed at varying times after injection and processed for electron microscopy by conventional techniques. Cells fixed 1–3 min after injection contained HRP reaction product only in the cell body. HRP reaction product was found at varying distances down axons in direct relation to the interval between injection and fixation. Colloidal gold particles were found throughout the cell body but not in axons of tissue fixed 1–3 min after injection. Both HRP reaction product and 5-nm colloidal gold particles were observed within the microvillar projections of internal and external rhabdomere, as well as within the extracisternal spaces of endoplasmic reticulum. We conclude that large molecules injected from an intracellular micropipette into an arbitrary locus of ventral photoreceptor cells have access to all of the presumed sites of the phototransduction cascade.

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.

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.

Photoelectron imaging is a novel way of imaging the distribution of specific cell surface components. The basic principle is the photoelectric effect, in which electrons are ejected from the specimen by UV light. There are two primary forms of image contrast in photoelectron microscopy: material contrast and topographical contrast. Material contrast, provided by differences in photoelectron quantum yields, makes it possible to select labels that appear bright against the darker background of the cell surface. Topographical contrast ensures that label distribution can be interpreted in the context of cellular structures. By varying the wavelength of the exciting light, the contrast between label and cell surface can be controlled (tuned) to produce images ranging from a primarily topographical view of the surface to those showing only the label distribution. Being an emission-based technique, photoelectron imaging shares with fluorescence microscopy the ability to image label distributions at low magnification. However, unlike fluorescence microscopy the photoelectron image is formed by electrons, which generates a greater level of attainable resolution. Label contrast and wavelength-dependent tunability of contrast is illustrated with images of silver-enhanced immunogold-labeled cell surfaces, including selective labeling of cells in mixed-cell co-cultures, the cell surface CD44 adhesion protein on human glioma cells, fibronectin patterns on human fibroblasts, and the human transferrin receptor on MCF-7 human breast carcinoma cells.