The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Mid-infrared or Raman spectroscopy together with multivariate data analysis provides a novel approach to clinical laboratory analysis, offering benefits due to its reagent-free nature, the speed of the analysis and the possibility of obtaining a variety of information from one single measurement. We compared mid-infrared and Raman spectra of the sera obtained from 247 blood donors. Partial least squares analysis of the vibrational spectra allowed for the quantification of total protein, cholesterol, high and low density lipoproteins, triglycerides, glucose, urea and uric acid. Glucose (mean concentration: 154 mg/dl) is frequently used as a benchmark for spectroscopic analysis and we achieved a root mean square error of prediction of 14.7 and 17.1 mg/dl for mid-infrared and Raman spectroscopy, respectively. Using the same sample set, comparable sample throughput, and identical mathematical quantification procedures Raman and mid-infrared spectroscopy of serum deliver similar accuracies for the quantification of the analytes under investigation. In our experiments vibrational spectroscopy-based quantification appears to be limited to accuracies in the 0.1 mmol/l range.

The fast and reliable determination of concentrations of blood, plasma or serum constituents is a major requirement in clinical chemistry. We explored Raman spectroscopy as a reagent-free tool for predicting the concentrations of different parameters in serum and serum ultrafiltrate. In an investigation using samples from 247 blood donors (which we believe to be the largest study on Raman spectroscopy of serum so far) the concentrations of glucose, triglycerides, urea, total protein, cholesterol, high density lipoprotein, low density lipoprotein and uric acid were determined with an accuracy within the clinically interesting range. After training a multivariate algorithm for data analysis, using 148 samples, concentrations were predicted blindly for the remaining 99 serum samples based solely on the Raman spectra. Relative errors of prediction around 12% were obtained. Moreover, to the best of our knowledge, differentiation between HDL and LDL cholesterol as well as the quantification of uric acid was for the first time successfully accomplished for serum-based Raman spectroscopy. Finally, we showed that ultrafiltration can efficiently reduce fluorescent light background to improve prediction accuracy such that the relative coefficient of variation was reduced for glucose and urea in ultrafiltrate by more than a factor of 2 when compared to serum.