Polarimetric optical-fibre sensor for biochemical measurements

The use of an optical-fibre polarimeter as a chemical sensor is demonstrated. The compound to be detected is allowed to adsorb onto a decladded 5 cm length of the fibre. The fibre is polarization maintaining with an elliptical fibre core and a D-shaped geometry. The overall retardation stability of this fibre polarimeter is ≈ 0.5 × 2π rad m−1 K−1. With this sensor adsorption processes of proteins can be followed on-line. The resulting relative phase retardations caused by the growth of a monolayer of antibodies (αhCG, αhSA) are 0.25 × 2π. For the much smaller protein hSA, this value is 0.1 × 2

Polarization-sensitive resonance CSRS of deoxy-and oxyhaemoglobin

Polarization-sensitive coherent Stokes Raman scattering (CSRS) measurements of oxy- and deoxyhaemoglobin in aqueous solutions are reported. The excitation wavelengths used were chosen in the region of the Q absorption bands to achieve twofold electronic resonance. The dispersion profiles of all independent susceptibility (3) components and purely anisotropic and anti-symmetric scattering contributions were resolved within the frequency non-degenerate CSRS scheme. Eight bands of oxyhaemoglobin and five bands of deoxyhaemoglobin were observed in the range 1500-1680 cm-1. Simultaneously fitting sets of polarization spectra provided vibrational parameters (positions, bandwidths, amplitudes, phases and CSRS depolarization ratios) for each compound. Major bands were assigned to the non-totally symmetric v10, v11 and v19 modes of the porphyrin macrocycle. The phases calculated exhibited a correlation with the symmetry of the vibrations. On the basis of the spectral fits, the three additional peaks arising in the oxyhaemoglobin spectra could be ascribed to the bands of intermediate deoxyhaemoglobin. The occurrence is due to the partial photolysis of oxyhaemoglobin. Vibrational parameters of these bands were found to be essentially similar to the parameters of the bands observed in the spectra of the stable deoxyhaemoglobin. Despite the asymmetric character predicted, the major bands were all contributed to by a considerable isotropic component. A decrease in the depolarization ratio PR1212 of the anomalously polarized v19 mode from 7.7 in oxyhaemoglobin to 4.3 in deoxyhaemoglobin was observed. Such a decrease in anti-symmetric character of the vibration on release of the ligand supports the occurrence of deformation of the haem ring system

Cluster analysis of flow cytometric list mode data on a personal computer

A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce the large number of subclusters. Thus we combine the advantage of the k-means (speed) with that of the nearest neighbor technique (accuracy). In order to achieve a rapid analysis, no complex data transformations such as principal components analysis were used. \ud Results of the cluster analysis on both real and artificial flow cytometric data are presented and discussed. The results show that it is possible to get very good cluster analysis partitions, which compare favorably with manually gated analysis in both time and in reliability, using a personal computer

A new principle of cell sorting by using selective electroporation in a modified flow cytometer

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. \ud We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 s and resulting field strengths of 2.0 and 3.2 X 106 V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). \ud The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%

Resonance polarization and phase-mismatched CARS of pheophytin b excited in the Qy band

Resonance polarization and phase-mismatched coherent anti-Stokes Raman scattering (CARS) measurements were performed on pheophytin b dissolved in acetone excited in the Qy absorption band, where strong broad fluorescence makes spontaneous Raman spectroscopy impossible. The phase-mismatching technique was applied to suppress solvent background and used in combination with the polarization-sensitive CARS technique to measure directly the x1111(3) and x1221(3) components to estimate depolarization ratios. The spectra were fitted by a non-linear least-squares procedure yielding vibrational band parameters. Some CARS dispersion information on the vibrational amplitudes was obtained by varying the pump wavelength. CARS excitation profiles based on transform theory were calculated and partly explain the observed amplitude dispersion. The application of the combined phase-mismatched polarization CARS technique may be useful in many other cases of highly fluorescing molecules when resonantly excited

Thickness and dielectric constant determination of thin dielectric layers

We derive a method for the determination of the dielectric constant and thickness of a thin dielectric layer, deposited on top of a thick dielectric layer which is in turn present on a metal film. Reflection of p- and s-polarized light from the metal layer yields minima for certain angles of incidence where the light is absorbed by the metal. The thin dielectric layer causes shifts in the angles at which the minima occur, from which the thickness and dielectric constant can be obtained. The model is tested for 3.5 and 14 nm thick photoresist gratings

Secondary structure of bovine albumin as studied by polarization-sensitive multiplex CARS spectroscopy

The first application of polarization-sensitive multiplex coherent anti-Stokes Raman spectroscopy (MCARS) in the absence of resonance enhancement to the resolution of the secondary structure of a protein in solution is reported. Polarization MCARS spectra of bovine albumin in D2O were obtained in the range 1370 to 1730 cm−1 with the aid of the background suppression technique. The spectra were fitted simultaneously with a single set of parameters (band positions, bandwidths, amplitudes, and depolarization ratios). Polarized Raman spectra simulated with these parameters revealed a good correspondence with the spontaneous Raman spectra measured. The broad amide I′ band was decomposed assuming the three major secondary conformations of protein, of which the contribution of β-sheet structure was found to be negligible. Relative weights of α-helix and random coil conformations agree well with the estimates obtained with Raman and circular dichroism (CD) spectroscopies

Local variation in absolute water content of human and rabbit eye lenses measured by Raman microspectroscopy

Raman spectra were obtained from fresh, fixed and sliced rabbit lenses and from human lens slices. For all lenses and lens slices the ratio R, defined as the Raman intensity at 3390 cm−1 divided by the Raman intensity at 2935 cm−1, was measured at different locations along the visual and equatorial axis. The ratios R were transformed to absolute water mass percentages by measuring solutions with known protein concentrations. It was shown that fixation and slicing have very little effect on the absolute water content of the lenses. The values obtained for the absolute water content are comparable to values given in literature. It was also shown that the water content in rabbit and human lenses rapidly decreases from the immediate anterior and posterior subsurface region to the deep superficial cortex and is relatively constant in the nucleus. Raman microspectroscopy appears to be a reliable method for the measurement of the absolute water content of small volumes on defined positions in the lens. This can be very useful when analyzing the possible relation between local variations in water content and the occurrence of opacities in the lens