Ambient optical detection of labeled molecules is limited for fluorescent dyes by photobleaching and for semiconducting nanoparticles by “blinking” effects. Because nanometer-sized metal particles do not optically bleach, they may be useful optical labels if suitable detection signals can be found. We demonstrate far-field optical detection of gold colloids down to diameters of 2.5 nanometers with a photothermal method that combines high-frequency modulation and polarization interference contrast. The photothermal image is immune to the effects of scattering background, which limits particle imaging through Rayleigh scattering to diameters larger than 40 nanometers.

https://science.sciencemag.org/content/sci/297/5584/1160.full.pdf

Photothermal imaging of nanometer-sized metal particles among scatterers.

The CA1 pyramidal neurons in the hippocampus are selectively vulnerable to transient ischemic damage. In experimental animals, the CA1 pyramidal neurons undergo cell death several days after brief forebrain ischemia. It remains, however, unknown whether this delayed neuronal death is necrosis or apoptosis. To investigate the degenerating processes of the CA1 pyramidal neurons in gerbil hippocampus after brief ischemia, lysosomal and nuclear alterations in the cells were examined using immunocytochemistry, in situ nick-end labeling, and Southern blotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L, representative lysosomal cysteine proteinases, increased in the CA1 pyramidal neurons 3 d after ischemic insult, which showed cell shrinkage. By morphometric analysis, the volume density of cathepsin B-positive lysosomes markedly increased 3 d after ischemic insult, while that of autophagic vacuole-like structures also increased at this stage, suggesting that cathepsin B-immunopositive lysosomes increasing in the neurons after ischemic insult are mostly autolysosomes. Nuclei of the CA1 neurons were nick-end labeled by biotinylated dUTP mediated by terminal deoxytransferase 3 and 4 d after ischemic insult, but not in the prior stages. Simultaneously, dense chromatin masses appeared in nuclei of the neurons. By Southern blotting, laddering of DNA occurred only in CA1 hippocampal tissues obtained 4 d after ischemic insult. Confocal laser scanning microscopy demonstrated that the fragmented DNA in the CA1 pyramidal layer was phagocytosed by microglial cells. The results suggest that delayed death of the CA1 pyramidal neurons after brief ischemia is not necrotic but apoptotic.

https://www.jneurosci.org/content/jneuro/15/2/1001.full.pdf

Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis

Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.

/pdf/immuno-localization-of-the-insulin-regulatable-glucose-33gv1b6w07.pdf

Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or "cargoes." Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.

/pdf/nuclear-pore-complex-is-able-to-transport-macromolecules-2hy9xokni0.pdf

J W Slot

Papers

A new method of preparing gold probes for multiple-labeling cytochemistry