An improved optical method for surface plasmon resonance experiments

In this paper an inexpensive optical device is described, which is capable of measuring the optical reflectance at different angles, while keeping the laser spot stationary at one point of the surface. This is accomplished by applying cylindrical optics. Its use is demonstrated in a surface plasmon resonance (SPR) sensor. A coil-operated vibrating mirror is used to obtain an angle scan of about 4 degrees. The angle of minimum reflectance can be detected with an accuracy of approximately 2 × 10−3 degrees. Despite the use of simple optical components, disturbance of laser beam parallelism is no more than 0.02 degrees. Displacement of the laser spot at the surface during the angle scan is kept within 0.2 mm. The device eliminates disturbances due to surface irregularities in measurements.\u

Detection of EpCAM negative circulating tumor cells in CellSearch waste

Introduction: Circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. The CellSearch system uses immunomagnetic enrichment targeting the EpCAM antigen. A frequently raised question is what the frequency and significance is of CTC that do not express EpCAM within the CTC of the individual patient and between different patients. To investigate this, a device was constructed that collects the blood discarded by the CellSearch system and passes the blood through micro sieves with 5um pores to enrich for the larger CTC. Methods: A sample collection device was attached to the waste line of the CellTracks Autoprep (AP). The blood discarded after immunomagnetic separation was detected in the waste line and collected into a separate 50ml conical tube for each patient sample. After collection, the blood was filtered through microfabricated silicon nitride filters with pore diameters of 5 micrometer (Aquamarijn, Zutphen, The Netherlands) with a pressure below 40mbar. To evaluate recovery on the filter the COLO320(median size 11μm), SKBR3(16μm) and T24(16μm) cell lines where used. To evaluate recovery from the AP waste the SKBR3(445000 EpCAM antigens) and T24(2167 EpCAM antigens) cell lines where used. 300 cells where spiked in 7.5ml of blood collected in CellSave tubes from healthy volunteers. All samples were processed the day after collection. Results: The average recovery on filter from whole blood is 29% for COLO320 and ≥80% for SKBR3 and T24. The average recovery of the SKBR3 cell line with the CellSearch system is 91% and 5% are recovered from the AP waste using filtration. In contrast, the average recovery of T24 cell line with the CellSearch system is only 30% but 51% are recovered from the AP waste after filtration. The carryover between spiked and unspiked samples collected from the AP waste was <1.3% of the spiked cells for unspiked samples with a mean of 0.9%. Conclusions: We present a method and device that enables the identification and characterization of CTC not detected by the CellSearch system which allows a systematic evaluation of the clinical relevance of these CTC

Oxidative stabilization of mixed mayonnaises made with linseed oil and saturated medium-chain triglyceride oil

Mayonnaises, made with either saturated medium chain triglyceride (MCT) oil or unsaturated purified linseed oil (LSO), were mixed. Raman confocal microspectrometry demonstrated that lipid droplets in mixed mayonnaise remained intact containing either MCT oil or LSO. Peroxide formation during storage was lower in mixed mayonnaise compared to LSO mayonnaise, while in mixed oil mayonnaise the level of peroxides was constantly low. Mixed oil mayonnaise had a lower rate of oxygen consumption than mixed mayonnaise, LSO mayonnaise having the highest rate. The decay of water-soluble nitroxyl radicals showed radicals are formed in the aqueous phase with the same rate independent of the lipids. This was also reflected in decay of α-tocopherol during storage being similar in MCT and LSO mayonnaises, but being stable in mixed oil mayonnaise and mixed mayonnaise. Results suggest that other effects than simply diluting unsaturated triglycerides with saturated triglycerides is causing the oxidative stabilization observed for mixed mayonnaise and mixed oil mayonnais

A microfluidic chip for high resolution Raman imaging of biological cells

A microfluidic chip was designed, prepared and tested for integration with a confocal Raman imaging spectrometer with the specific purpose of enabling studies of individual biological cells. The design of the chip effectively overcomes the limitations arising from the high numerical aperture (NA) and short working distance objectives, which are necessary for high resolution imaging. The high confocal spatial resolution was achieved by a careful design of the geometry of the chip together with a thin, optically and Raman silent sealing window as the embedding medium for the channels. A leak-free microfluidic chip was obtained by surface plasma modification of polydimethylsiloxane (PDMS) and optimization of the liquid loading parameters. Raman images of biological cells, which were transported by flow into the microfluidic chip, are presented as an example of a Raman-microfluidics application. The broad band Raman spectra from −50 cm−1 to 3650 cm−1 were recorded in 1600 frequency intervals without any signal enhancement or sample labeling. Raman images were recorded in ∼400 seconds and they typically consisted of 64 × 64 pixels with a step size of 250 nm, thus containing ∼4 × 106 data points altogether

Protein profiles in cortical and nuclear regions of aged human donor lenses: A confocal Raman microspectroscopic and imaging study

A combination of Raman spectroscopy, imaging, hierarchical cluster analysis (HCA) and peak ratio analysis was used to analyze protein profiles in the superficial cortex (SC), deep cortex (DC) and nucleus of old human lenses with cortical, nuclear and mixed cataracts. No consistent differences were observed in protein spectra and after cluster analysis between the three locations irrespective of the presence or absence of cortical opacities and/or coloration. A sharp increase (∼15%–∼33%) in protein content from SC to DC, normal for human lenses, was found in 7 lenses. In 4 lenses, characterized by the absence of cortical opacities, the SC has a protein content of ∼35%. A significant increase in the disulfide-to-protein ratio is found only in the SC of the 7 cortical cataracts. No changes were found in sulfhydryl-to-protein ratio. The relative contents of α-helices and β-sheets increase from SC to nucleus. β-Sheets are more common in the SC of lenses with cortical cataract. The absence of significant and consistent changes in protein profiles between nucleus and cortex even in cases of severe coloration is not favoring the prevailing concept that ubiquitous protein oxidation is a key factor for age related nuclear (ARN) cataracts. The observations favor the idea that multilamellar bodies or protein aggregates at very low volume densities are responsible for the rise in Mie light scatter as a main cause of ARN cataracts leaving the short-range-order of the fiber cytoplasm largely intact. The absence of significant changes in the protein spectra of the deep cortical opacities, milky white as a result of the presence of vesicle-like features, indicate they are packed with relatively undisturbed crystallins

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides