Electric field standing wave effects in FT-IR transflection spectra of biological tissue sections: Simulated models of experimental variability

The so-called electric field standing wave effect (EFSW) has recently been demonstrated to significantly distort FT-IR spectra acquired in a transflection mode, both experimentally and in simulated models, bringing into question the appropriateness of the technique for sample characterization, particularly in the field of spectroscopy of biological materials. The predicted effects are most notable in the regime where the sample thickness is comparable to the source wavelength. In this work, the model is extended to sample thicknesses more representative of biological tissue sections and to include typical experimental factors which are demonstrated to reduce the predicted effects. These include integration over the range of incidence angles, varying degrees of coherence of the source and inhomogeneities in sample thickness. The latter was found to have the strongest effect on the spectral distortions and, with inhomogeneities as low as 10% of the sample thickness, the predicted distortions due to the standing wave effect are almost completely averaged out. As the majority of samples for biospectroscopy are prepared by cutting a cross section of tissue resulting in a high degree of thickness variation, this finding suggests that the standing wave effect should be a minor distortion in FT-IR spectroscopy of tissues. The study has important implications not only in optimization of protocols for future studies, but notably for the validity of the extensive studies which have been performed to date on tissue samples in the transflection geometry.     

Electric field standing wave effects in FT-IR transflection spectra of biological tissue sections: Simulated models of experimental variability

Published:The latter was found to have the strongest effect on the spectral distortions and, with inhomogeneities as low as 10% of the sample thickness, the predicted distortions due to the standing wave effect are almost completely averaged out.Corrected:The latter two were found to have the strongest effect on the spectral distortions and, with inhomogeneities as low as 10% of the sample thickness, the predicted distortions are almost completely averaged out, while with a completely incoherent incident light, the effect of the standing wave is completely absent.

4. Averaging over phase

Published:Similar to the case of angle of incidence, an averaging effect is apparent. The overall amplitude changes are smaller, although, for the peak close to 1000 cm⁻¹, an increase is seen. Nevertheless, the effect of considering a distribution of incident wave phases is a reduction of effect on the EFSW.Corrected:It is clear that, when taking into consideration completely incoherent light interacting with the sample, the EFSW distortion is completely averaged out. The same result was obtained for all other thicknesses.

6. A combination effect

Published:The incorporation of all three parameters at once even further improves the simulated spectrum of a hypothetical absorber. The combination effect was also calculated for other thickness ranges of interest and is shown in Fig. 9.Again the mean spectra of samples in the range > 2 μm are very close to the original hypothetical absorber. The spectral range most variable is the one associated with longest wavelength, i.e. close to 1000 cm⁻¹, due to inherently fewer nodes at this wavelength penetrating the sample.Corrected:The incorporation of all three parameters at once retrieves the simulated spectrum of a hypothetical absorber. The combination effect was also calculated for other thickness ranges of interest and is shown in Fig. 5, Fig. 8, Fig. 9.

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Fig. 5. Simulated spectra of three phases out of 18 different phases Δ from 0 to 2π and their mean (blue). (For interpretation of the references to color in this sentence, the reader is referred to the web version of the article.)

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Fig. 8. A combination effect taking into account all three parameters (incidence angle, phase and thickness) at once compared with a spectrum averaged over thickness only.

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Fig. 9. A combination effect of EFSW averaging for different thicknesses ranges.

7. Final discussion

Published:Out of the three parameters considered, averaging of thickness inhomogeneities was found to have the most prominent impact on the effects of EFSW on the modeled absorption spectrum. The averaging renders the EFSW negligible for samples with higher thickness. It is important to note, however, that the low thickness regime (<2 micrometers) is less influenced by the averaging effects.Corrected:Out of the three parameters considered, averaging of thickness inhomogeneities and light incoherence were found to have the most prominent impact on the effects of EFSW on the modeled absorption spectrum. In both cases, the averaging renders the EFSW negligible. It is important to note, however, that the low thickness regime (<2 micrometers) is less influenced by the inhomogeneity averaging effect.

8. Conclusions

Published:Out of three potential averaging effects identified here, the thickness averaging can play a pivotal role in significantly lowering the EFSW in transflection FT-IR spectra, when the lateral length scale of the spatial variations are of similar dimensions to the spatial resolution of the measurement. This is a case for imaging of many tissue cross-sections, but maybe not relevant when in vitro single cells are measured. Angle and phase averaging also have a significant effect on the EFSW and along with thickness averaging for samples above 2 μm of thickness, the EFSW is almost completely removed.Corrected:Out of three potential averaging effects identified here, the thickness averaging can play a pivotal role in significantly lowering the EFSW in transflection FT-IR spectra, when the lateral length scale of the spatial variations are of similar dimensions to the spatial resolution of the measurement. This is a case for imaging of many tissue cross-sections, but maybe not relevant when in vitro single cells are measured. A complete light incoherence leads to removal of EFSW effect. Angle averaging also has an effect on the EFSW and along with the other two effects, the result is that the EFSW is completely removed.     

Electric field induced structure formation in free standing ferroelectric films

Free standing ferroelectric films in a rotating electric field have been investigated. Depending upon the applied voltage and the period of the field rotation different structure formations could be observed. At high fields, a homogeneous orientation is obtained. In the case of very low fields, a schlieren texture results. Under special conditions, a stable ring system appears. The dynamics of these rings have been studied. The dependence of the structure formation on the relation between the impulse length of the applied field and the reorientation time is discussed.     

Electric field induced structure formation in free standing ferroelectric films

Abstract

Free standing ferroelectric films in a rotating electric field have been investigated. Depending upon the applied voltage and the period of the field rotation different structure formations could be observed. At high fields, a homogeneous orientation is obtained. In the case of very low fields, a schlieren texture results. Under special conditions, a stable ring system appears. The dynamics of these rings have been studied. The dependence of the structure formation on the relation between the impulse length of the applied field and the reorientation time is discussed.     

Thermal analysis of the melting process of poly(trimethylene terephthalate) using FTIR micro-spectroscopy

Thermal spectra of poly(trimethylene terephthalate) (PTT) were collected over a temperature range of 40-250 °C by FTIR micro-spectroscopy. Based on the changes of absorbance ratio corresponding to characteristic groups in low and high vibration energy states, the apparent enthalpy differences of vibration energy states transformation (ΔH_v) in the melting process have been calculated by van’t Hoff equation at constant pressure. In comparison with the values of ΔH_v, the status of participation for the vibration mode of various characteristic groups in PTT macromolecular chain segments was analyzed. It was found that the vibration modes related to the trimethylene glycol unit (O-CH₂-CH₂-CH₂-O) of PTT behaved significant sensitivity and made prominent contribution in the melting process. By the summarization of corresponding data, it has shown that the melting course concerned amorphous phase began at as early as 218 °C, accompanied by the occurrence of crystallization to certain extent, and the ending point was at approximately 238 °C; whereas the melting course concerned crystalline phase began till 228 °C, with the top value of 238 °C, and ended at 242 °C. Besides, for the particular ordered arrangement of chain segments of aromatic polyesters in the melting course, FTIR analysis has provided a reasonable explanation on a molecular level.     

Electric field effects on director pattern and disclinations in free standing nematic films of 8CB

Studies of electric field-induced orientational effects are carried out on substrate-free nematic 8CB films, held between parallel electrodes and containing a peripheral-1/2 loop. The loop separates the birefringent boundary from the central homeotropic zone. Static and dynamic characteristics of the splay-bend layers contiguous to the electrodes are investigated by polarizing microscopy. A model based on director field symmetry is proposed to explain (a) the growth of the transition layers independently of the associated singularities and (b) the formation or otherwise of static solitons (Brochard-Leger walls). Linear dependence of the growth rate of transition layers on field strength is compared with a similar behaviour for line defects. Bifurcation of a soliton at elevated voltages into a pair of - 1/2 lines, through a non-pincement process, is described.     

Electric field effects on director pattern and disclinations in free standing nematic films of 8CB

Studies of electric field-induced orientational effects are carried out on substrate-free nematic 8CB films, held between parallel electrodes and containing a peripheral-1/2 loop. The loop separates the birefringent boundary from the central homeotropic zone. Static and dynamic characteristics of the splay-bend layers contiguous to the electrodes are investigated by polarizing microscopy. A model based on director field symmetry is proposed to explain (a) the growth of the transition layers independently of the associated singularities and (b) the formation or otherwise of static solitons (Brochard-Leger walls). Linear dependence of the growth rate of transition layers on field strength is compared with a similar behaviour for line defects. Bifurcation of a soliton at elevated voltages into a pair of - 1/2 lines, through a non-pincement process, is described.     

Optical artefacts in transflection mode FTIR microspectroscopic images of single cells on a biological support: the effect of back-scattering into collection optics

Infrared microspectroscopic imaging data of single human prostate cancer cells, on an artificial extracellular matrix (Matrigel) thin-film surface, are presented. The spectral intensity maps, obtained in reflection mode, appear to show that the protein intensity distribution observed at the location of a cell changes dramatically depending on the concentration and/or thickness of the underlying Matrigel layer. Specifically, cells adhered to a low protein concentration or thin surface exhibit a higher protein intensity signal than the surrounding layer whereas those on a high protein concentration or thick surface exhibit a lower protein intensity signal. These results are qualitatively explained by a simple model that takes into account the fact that radiation scattered from cells can enter the collection optics of the microscope without passing through the Matrigel layer. This leads to an apparent reduction in absorption at the cell.     

Effect of energy dependence of primary beam divergence on the X-ray standing wave characterization of layered materials.

Incident primary beam divergence is a source of systematic error in X-ray standing wave (XSW) characterization of single and multilayer thin films. Primary beam divergence significantly alters the XSW profile of a layered material and can lead to large errors when used with higher excitation energies. The present study suggests that when one uses Mo-Kalpha excitation, the primary beam divergence should be in range of 0.005(0). On the other hand, in the case of Cu-Kalpha excitation, primary beam divergence can be relaxed up to 0.01(0).     

FTIR spectroscopic study of titanium-containing mesoporous silicate materials

The surface acidity of different mesoporous titanium-silicates, such as well-organized hexagonally packed Ti-MMM, Ti-MMM-2, Ti-SBA-15, and amorphous TiO(2)-SiO(2) mixed oxides (aerogels and xerogels), was studied by means of FTIR spectroscopy of CO adsorbed at 80 K and CD(3)CN adsorbed at 293 K. The surface hydroxyl groups of mesoporous titanium-silicates with 2-7 wt % Ti revealed a Br?nsted acidity slightly higher to that of pure silicate. TiO(2)-SiO(2) xerogels revealed the highest Br?nsted acidity among the titanium-silicates studied. CO adsorption revealed two additional sites on the surface in comparison to pure silicate, characterized by nu(CO) from 2185 (high pressure) to 2178 (low pressure) cm(-1) and from 2174 (high pressure) to 2170 (low pressure) cm(-1). These bands are due to CO adsorbed on isolated titanium cations in the silica surrounding or having one Ti(4+) cation in their second coordination sphere and due to CO interactions with Ti-OH groups, respectively. CD(3)CN adsorption similarly revealed the existence of two additional sites, which were not detected for pure silicate: at 2289 cm(-1) due to CD(3)CN interaction with titanol groups and from 2306 (low pressure) to 2300 (high pressure) cm(-1) due to acetonitrile interaction with isolated framework titanium cations with probably one Ti(4+) cation in their second coordination shell. The spectroscopic results are compared with computational data obtained on cluster models of titanium-silicate with different titanium content. According to the IR data, the Ti accessibility on the surfaces for mesoporous titanium-silicates with similar Ti loading (2 wt %) was found to fall in the order TiO(2)-SiO(2) aerogel approximately TiO(2)-SiO(2) xerogel > Ti-MMM approximately Ti-MMM-2 > Ti-SBA-15. This order (except TiO(2)-SiO(2) xerogel) correlates with the catalytic activity found previously for titanium-silicates in 2,3,6-trimethylphenol oxidation with H(2)O(2).     

Phosphorus determination in biological materials

Summary An analysis scheme for the spectrophotometric determination of phosphorus as phosphate in biological materials has been worked out. The wet digestion of the sample was carried out in Kjeldahl flasks by a mixture of perchloric and nitric acids. The relative standard deviation (RSD) of the obtained results is not more than 1% for amounts up to 30 g. The method was applied to various biological materials.     

Accuracy in analyses of biological materials

Summary The work of the Community Bureau of Reference is outlined, special emphasis being laid on the problem of accuracy. Examples are described for trace elements in milk, Pb and Cd in blood, Ca in serum, cortisol in serum and aflatoxin in milk.     

NBS activities in biological reference materials

Summary NBS activities in biological reference materials during 1986–1988 are described with a preview of plans for future certifications of reference materials. During the period, work has been completed or partially completed on about 40 reference materials of importance to health, nutrition, and environmental quality. Some of the reference materials that have been completed during the period and are described include: creatinine (SRM 914a), bovine serum albumin (SRM 927a), cholesterol in human serum (SRM's 1951–1952), aspartate aminotransferase (RM 8430), cholesterol and fat-soluble vitamins in coconut oil (SRM 1563), wheat flour (SRM 1567a), rice flour (SRM 1568a), mixed diet (RM 8431a), dinitropyrene isomers and 1-nitropyrene (SRM 1596), and complex PAH's from coal tar (SRM 1597). Oyster tissue (SRM 1566a) is being analyzed and should be available in 1988.

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NBS-Aktivitäten in biologischen Referenzmaterialien

     

Electric field spectrum of pyridazine

An electric field induced spectrum has been obtained in the region of the 0⁰₀ band of pyridazine near 3752 Å. Since all intensity in this single-peaked spectrum is negative we interpret this to indicate field induced mixing with a very dense set of states.     

Analyses of hazardous substances in biological materials

Summary The interest of the Commission for the Investigation of Health Hazards of Chemical Compounds in Work Area of the German Science Foundation in practical, sensitive and specific methods for monitoring the limit values led to the establishment of the working group Analytical Chemistry in 1969. A loose-leaf collection Analysen in biologischem Material and 3 volumes of an English edition have since been published. In the meantime the German edition of Analyses in biological materials has been supplemented ten times, including 100 methods for the measurement of about 170 parameters; the English edition is in 3 volumes with 45 selected methods. The volumes include only standard operational procedures that are suitable for routine use. The reliability of the analytical procedures is defined and checked for these reasons and because they satisfy the requirements of statistical quality control. The analytical methods published by the Working Group Analytical Chemistry are recommended by pertinent guidelines and regulations for use in Germany.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Quantitative determination of americium in biological materials

A simple, economic and specific radiochemical method for the determination of americium in urine and other biological materials is given. The urine sample is heated in nitric acid to break down any organic complexes of actinides. Insoluble phosphates are precipitated and changed to carbonates. The carbonate solution is passed through a diglycol succinate column and americium is eluted by hydrochloric acid. The eluate is evaporated to prepare a source for counting. The proposed procedure produces high overall yield and the sensitivity of the method is about 0.74 mBq (0.02 pCi). Determination of americium in the presence of other -emitters in urine is also given.     

Electric pulse-induced precipitation of biological macromolecules in electroporation

We found that electric discharge through solution of biological macromolecules (DNA, RNA and proteins) causes precipitation of significant portions of these macromolecules. This precipitation is a consequence of the interaction of biological macromolecules with the metal ions solubilized from the anode plate by the electric pulse, and occurs in both absence and presence of the cells in poration medium. Precipitated fractions of macromolecules sediments at the centrifugation speed used to pellet eukaryotic cells and does not dissolve when washed with buffer. Our data indicate a complication of the direct evaluation of electroporation efficiency based on the assumption that electroporated biological macromolecules which remain associated with the cells after several washes, are successfully electroinjected into the cytoplasm of cells.     

Electric field effects in proteins in membranes

Both the organization and function of protein nanostructures in membranes are related to the substructural properties of the lipid portion of the membrane. Potential differences that are established across the membrane and generate electric fields in these very thin portions are shown to modulate the organizational and functional properties of the protein modules. Many protein modules also have nonisotropic distributions of charged sites, including configurations in which there are regions containing predominantly positive fixed charges, juxtaposed with adjacent regions containing predominantly negative fixed charges. In these double fixed charge regions, very large electric fields can manifest in the ionic depletion layer at the junction of the two fixed charge regions.Consideration is also given to the manner in which the intense electric fields that are established in protein modules, such as proton ATPases, can modulate the chemical reactions that are associated with proton transport and dehydration reactions.