Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80u thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters

Quantifying the Reversible Association of Thermosensitive Nanoparticles

Under many conditions, biomolecules and nanoparticles associate by means of attractive bonds, due to hydrophobic attraction. Extracting the microscopic association or dissociation rates from experimental data is complicated by the dissociation events and by the sensitivity of the binding force to temperature (T). Here we introduce a theoretical model that combined with light-scattering experiments allows us to quantify these rates and the reversible binding energy as a function of T. We apply this method to the reversible aggregation of thermoresponsive polystyrene/poly(N-isopropylacrylamide) core-shell nanoparticles, as a model system for biomolecules. We find that the binding energy changes sharply with T, and relate this remarkable switchable behavior to the hydrophobic-hydrophilic transition of the thermosensitive nanoparticles

Reproducibility of oligonucleotide arrays using small samples

BACKGROUND: Low RNA yields from small tissue samples can limit the use of oligonucleotide microarrays (Affymetrix GeneChips(®)). Methods using less cRNA for hybridization or amplifying the cRNA have been reported to reduce the number of transcripts detected, but the effect on realistic experiments designed to detect biological differences has not been analyzed. We systematically explore the effects of using different starting amounts of RNA on the ability to detect differential gene expression. RESULTS: The standard Affymetrix protocol can be used starting with only 2 micrograms of total RNA, with results equivalent to the recommended 10 micrograms. Biological variability is much greater than the technical variability introduced by this change. A simple amplification protocol described here can be used for samples as small as 0.1 micrograms of total RNA. This amplification protocol allows detection of a substantial fraction of the significant differences found using the standard protocol, despite an increase in variability and the 5' truncation of the transcripts, which prevents detection of a subset of genes. CONCLUSIONS: Biological differences in a typical experiment are much greater than differences resulting from technical manipulations in labeling and hybridization. The standard protocol works well with 2 micrograms of RNA, and with minor modifications could allow the use of samples as small as 1 micrograms. For smaller amounts of starting material, down to 0.1 micrograms RNA, differential gene expression can still be detected using the single cycle amplification protocol. Comparisons of groups of four arrays detect many more significant differences than comparisons of three arrays

Bias in the journal impact factor

The ISI journal impact factor (JIF) is based on a sample that may represent half the whole-of-life citations to some journals, but a small fraction (<10%) of the citations accruing to other journals. This disproportionate sampling means that the JIF provides a misleading indication of the true impact of journals, biased in favour of journals that have a rapid rather than a prolonged impact. Many journals exhibit a consistent pattern of citation accrual from year to year, so it may be possible to adjust the JIF to provide a more reliable indication of a journal's impact.Comment: 9 pages, 8 figures; one reference correcte

Development of a Large Field-of-View PIV System for Rotorcraft Testing in the 14- x 22-Foot Subsonic Tunnel

A Large Field-of-View Particle Image Velocimetry (LFPIV) system has been developed for rotor wake diagnostics in the 14-by 22-Foot Subsonic Tunnel. The system has been used to measure three components of velocity in a plane as large as 1.524 meters by 0.914 meters in both forward flight and hover tests. Overall, the system performance has exceeded design expectations in terms of accuracy and efficiency. Measurements synchronized with the rotor position during forward flight and hover tests have shown that the system is able to capture the complex interaction of the body and rotor wakes as well as basic details of the blade tip vortex at several wake ages. Measurements obtained with traditional techniques such as multi-hole pressure probes, Laser Doppler Velocimetry (LDV), and 2D Particle Image Velocimetry (PIV) show good agreement with LFPIV measurements