We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

Organelle transport along microtubules is believed to be mediated by organelle-associated force-generating molecules. Two classes of microtubule-based organelle motors have been identified: kinesin and cytoplasmic dynein. To correlate the mechanochemical basis of force generation with the in vivo behaviour of organelles, it is important to quantify the force needed to propel an organelle along microtubules and to determine the force generated by a single motor molecule. Measurements of force generation are possible under selected conditions in vitro, but are much more difficult using intact or reactivated cells. Here we combine a useful model system for the study of organelle transport, the giant amoeba Reticulomyxa, with a novel technique for the non-invasive manipulation of and force application to subcellular components, which is based on a gradient-force optical trap, also referred to as 'optical tweezers'. We demonstrate the feasibility of using controlled manipulation of actively translocating organelles to measure direct force. We have determined the force driving a single organelle along microtubules, allowing us to estimate the force generated by a single motor to be 2.6 x 10(-7) dynes.

We describe a rapid noncontact method for the capture of single cells or small tissue areas of any size or shape directly within the cap of a common microfuge tube. Prior to the laser-mediated transfer, the specimen is isolated by laser microbeam microdissection, forming a clear-cut gap around the selected area. Laser treatment does not impair subsequent RNA analysis. We have used this method to isolate a single cell from archival colon adenocarcinoma, and were able to detect point mutations within codon 12 of c-Ki-ras2 mRNA after nested RT-PCR analysis.

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Rapid identification of agronomically important genes is of pivotal interest for crop breeding. One source of such genes are crop wild relative (CWR) populations. Here we used a CWR population of <200 wild beets (B. vulgaris ssp. maritima), sampled in their natural habitat, to identify the sugar beet (Beta vulgaris ssp. vulgaris) resistance gene Rz2 with a modified version of mapping-by-sequencing (MBS). For that, we generated a draft genome sequence of the wild beet. Our results show the importance of preserving CWR in situ and demonstrate the great potential of CWR for rapid discovery of causal genes relevant for crop improvement. The candidate gene for Rz2 was identified by MBS and subsequently corroborated via RNA interference (RNAi). Rz2 encodes a CC-NB-LRR protein. Access to the DNA sequence of Rz2 opens the path to improvement of resistance towards rhizomania not only by marker-assisted breeding but also by genome editing.

To investigate interrelations of human obligate airway pathogens, such as Bordetella pertussis, and their hosts test systems with high in vitro/in vivo correlation are of urgent need. Using a tissue engineering approach, we generated a 3D test system of the airway mucosa with human tracheobronchial epithelial cells (hTEC) and fibroblasts seeded on a clinically implemented biological scaffold. To investigate if hTEC display tumour-specific characteristics we analysed Raman spectra of hTEC and the adenocarcinoma cell line Calu-3. To establish optimal conditions for infection studies, we treated human native airway mucosa segments with B. pertussis. Samples were processed for morphologic analysis. Whereas our test system consisting of differentiated epithelial cells and migrating fibroblasts shows high in vitro/in vivo correlation, hTEC seeded on the scaffold as monocultures did not resemble the in vivo situation. Differences in Raman spectra of hTEC and Calu-3 were identified in distinct wave number ranges between 720 and 1662 cm(-1) indicating that hTEC do not display tumour-specific characteristics. Infection of native tissue with B. pertussis led to cytoplasmic vacuoles, damaged mitochondria and destroyed epithelial cells. Our test system is suitable for infection studies with human obligate airway pathogens by mimicking the physiological microenvironment of the human airway mucosa.

Chromosome-specific paint probes provide a powerful tool with wide applications in cytogenetic analysis. Here, we present a new approach using UV-laser microbeam microdissection in combination with laser-pressure catapulting, which allows the fast isolation of single chromosomes for the generation of chromosome-specific paint probes. To demonstrate the feasibility of this approach, single chromosomes were collected and amplified with degenerate oligonucleotide-primed PCR, hapten-labeled and hybridized onto normal metaphase spreads. Fluorescence in situ hybridization signals revealed specific painting of the respective chromosomes.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.