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PubMed2006

Characterization of putative stem cell populations in the cornea using synchrotron infrared microspectroscopy.

PURPOSE: High-resolution synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy coupled with multivariate analysis was used to investigate the characteristics of putative adult stem cell (SC), transiently amplified (TA) cell, and terminally differentiated (TD) cell populations of the corneal epithelium. METHODS: Spectra of individual cells in situ in cryosections of bovine cornea were collected by using a synchrotron microspectroscopy facility at Daresbury Laboratory (United Kingdom). The resultant spectra were analyzed by multivariate analysis. RESULTS: The median spectra of the three different cell populations showed marked differences, which correlated with their degree of differentiation and proliferative capacity. Multivariate (principal component) analysis (PCA) showed that the three cell populations could be segregated into discrete clusters, with only a slight overlap between the SC and TA cell populations. Further analysis (Mann-Whitney test) indicated that the most significant (P<0.001) spectral differences between the SC and TA cell populations were chiefly associated with changes in nucleic acid conformation. CONCLUSIONS: SR-FTIR microspectroscopy coupled with PCA appears to enable the identification of SC, TA cell, and TD cell populations. The results also suggest that a small subpopulation of cells in the corneal epithelial SC niche possess TA cell-like characteristics. The most significant spectral characteristics associated with the SCs appear to relate to differences in nucleic acid conformation. This finding is consistent with recent theories suggesting that the control of differentiation is related to major changes in chromatin structure.

Animals↗
PubMed1996

Evaluation of sodium fluorescein for quantitative diagnosis of root caries.

The diagnosis of root caries, in particular the judgment of the activity of a visually observed lesion, is difficult. Quantitative determination of lesion severity would allow the lesion to be monitored with time, so that an indication of lesion activity could be obtained. This paper describes a step in the development of a method that provides such a quantitative determination. Specifically, fluorescein sodium salt is used as a penetrating dye, the subject of study being the relationship between dye concentration and porosity in demineralized root dentin. Fourteen human third molars were demineralized in vitro (lactic acid CMC-gel, pH 5; in each of 6 groups for 4, 7, 11, 14, 18, and 21 days). Fluorescein sodium salt (0.2 g/L) was applied for 2 min. Thin slices (+/- 130 microns) were cut from the root surfaces without water cooling. The dye fluorescence radiance in the demineralized dentin was determined by means of a micro-Raman spectroscope and compared with the mineral loss profiles measured with transverse microradiography (TMR). The TMR data were corrected for the difference in measurement area between the two measurement systems. Corrected TMR profiles were compared with the corresponding fluorescence scans, showing linear correspondence. The correlation coefficient was r = 0.96. We conclude that, after uptake of fluorescein sodium salt for 2 min, the dye concentration in an artificially produced root-surface caries lesion is proportional to the amount of mineral lost from that lesion.

Dentin↗
PubMed1997

Characterization of acidic vesicles in multidrug-resistant and sensitive cancer cells by acridine orange staining and confocal microspectrofluorometry.

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange. Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO. In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles. The resulting rise in the dye's oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry. In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 +/- 10%) than the value (48 +/- 8%) from K562-sensitive cells (p < 0.001). This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 microM), or nigericine (0.3 microM) all suppressed the initial difference of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one. The same effects were induced by 50 microM verapamil, a pleiotropic drug-resistance modulator. Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles.

Acridine Orange↗
PubMed1998

Confocal scanning microspectrofluorometry reveals specific anthracyline accumulation in cytoplasmic organelles of multidrug-resistant cancer cells.

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

Animals↗
PubMed2005

The influence of X-ray radiation on the mineral/organic matrix interaction of bone tissue: an FT-IR microscopic investigation.

Fourier transform infrared microscopy was used to investigate human cortical bone samples before and after treatment with increasing doses of X-ray radiation. Especially the spectral region of the v1 and v3 phosphate vibrations of hydroxyapatite, the main mineral component of bone, and the region of the amide I and amide II vibrational bands due to the collagen extracellular matrix were examined. Major spectral changes in the phosphate region between 1250-1000 cm(-1) occur after irradiation doses between 1 and 4 Gray. These findings are explained by a decrease in size of mineral crystallites and by variances of the toichiometric/non-stoichiometric apatite composition. The Ca2+ /PO4(3-) /HPO4(2-) composition in the biological apatite is altered near the bone surface. The secondary structure of the collagen matrix is not affected by cumulative irradiation up to doses of 15 Gray as indicated by the unchanged frequency maximum and contour shape of the amide I band between 1600-1700 cm(-1) . However, side chain carboxylate groups of the collagen matrix that are involved in coordination with apatite bound calcium ions are partially removed by decarboxylation upon irradiation. Concomitantly, a loss of acidic phosphate groups due to a formation of phosphate groups with bound calcium is observed. These changes on a molecular level can be correlated with alterations in the mechanical properties of the bone samples, e.g. with an increased embrittlement as deduced from experiments with a scanning acoustic microscope.

Amides↗
PubMed1992

Subcellular imaging of calcium exchange in cultured cells with ion microscopy.

Calcium to calcium exchange between intracellular and extracellular pools has been imaged directly in individual cells using stable 44Ca (98.78% enrichment) in the nutrient medium and an isotopic imaging technique, ion microscopy. Observations were made by imaging mass 40 to determine the native intracellular calcium (40Ca), and mass 44 to localize the 44Ca from the extracellular medium that exchanged with cellular calcium. LLC-PK1 porcine kidney epithelial cells were exposed to the nutrient medium with 1.87 mM stable 44Ca for 0, 1, 2, 5, 8, 20, 60 and 90 min, and cryogenically prepared prior to ion microscopic analysis. The cell nucleus, the Golgi region and the remaining cell cytoplasm could be spatially resolved to within about 0.5 microns using the ion microscope. On the basis of the kinetics of 40Ca to 44Ca exchange it was observed that all three compartments had a rapidly exchanging pool of calcium, which took about 2 min to exchange. A moderately rapidly exchanging pool of calcium was identified between 2 and 20 min of calcium exchange. The cells had exchanged about 50% of their total internal calcium with the external calcium in less than 20 min. The remaining 50% of the cellular calcium could be classified as a slowly exchanging pool. Isotopic images of 39K and 23Na were recorded along with 40Ca and 44Ca images to assess the health status of the cell. Isotopic imaging has the unique ability to distinguish intracellular calcium from the extracellular calcium that enters cells and has enormous potential for studies of calcium transport under physiological and pathological conditions.

Animals↗
PubMed2004

Pineal organs of deep-sea fish: photopigments and structure.

We have examined the morphology and photopigments of the pineal organs from a number of mesopelagic fish, including representatives of the hatchet fish (Sternoptychidae), scaly dragon-fish (Chauliodontidae) and bristlemouths (Gonostomidae). Although these fish were caught at depths of between 500 and 1000 m, the morphological organisation of their pineal organs is remarkably similar to that of surface-dwelling fish. Photoreceptor inner and outer segments protrude into the lumen of the pineal vesicle, and the outer segment is composed of a stack of up to 20 curved disks that form a cap-like cover over the inner segment. In all species, the pineal photopigment was spectrally distinct from the retinal rod pigment, with lambdamax displaced to longer wavelengths, between approximately 485 and 503 nm. We also investigated the pineal organ of the deep demersal eel, Synaphobranchus kaupi, caught at depths below 2000 m, which possesses a rod visual pigment with lambdamax at 478 nm, but the pineal pigment has lambdamax at approximately 515 nm. In one species of hatchet fish, Argyropelecus affinis, two spectral classes of pinealocyte were identified, both spectrally distinct from the retinal rod photopigment.

Animals↗
PubMed2005

Eyeshine and spectral tuning of long wavelength-sensitive rhodopsins: no evidence for red-sensitive photoreceptors among five Nymphalini butterfly species.

Spectral tuning of rhodopsins commonly refers to the effects of opsin amino acid substitutions on the wavelength for peak sensitivity of the rhodopsin absorption spectrum. Nymphalini butterflies provide an opportunity for identifying some of the amino acid substitutions responsible for insect rhodopsin spectral tuning because the majority of photoreceptor cells (R3-9) in the adult retina express only a single long wavelength-sensitive (LWS) opsin mRNA transcript. Therefore, the opsin genotype can be directly correlated with its phenotype. We determined the LWS opsin gene sequence from cDNA of the mourning cloak Nymphalis antiopa, and from genomic DNA of the malachite Siproeta stelenes and the peacock Inachis io. Using an epi-microspectrophotometer we examined each butterfly's eyeshine for photochemical evidence of multiple LWS rhodopsins and found only one. We then performed partial-bleaching experiments to obtain absorbance spectra for the LWS rhodopsins of all three species as well as from another nymphalid, the buckeye Junonia coenia. The isolated LWS opsin gene sequences varied in length from 1437-1612 bp and encode rhodopsins R522 (S. stelenes), R530 (I. io), R534 (N. antiopa) and, together with a previously published sequence, R510 (J. coenia). Comparative sequence analysis indicates that the S. stelenes rhodopsin is slightly blue-shifted compared to the typical 530 nm lepidopteran rhodopsin because of the presence of a S138A substitution at a homologous site that in mammalian MWS/LWS rhodopsins causes a 5 nm blue-shift. The difference in peak absorption between R522 of S. stelenes and R530 of Inachis io is therefore largely accounted for by this substitution. This suggests that spectral tuning mechanisms employing the S138A may have evolved in parallel in mammalian and butterfly MWS/LWS rhodopsins across 500 million years of evolution.

Amino Acid Sequence↗
PubMed1991

Photosensitive and photostable pigments in the retinae of Old World monkeys.

Microspectrophotometric measurements of retinal receptors are reported for eight species of Old World monkey. Although the animals vary greatly in size, colourings and habitat, they all appear to be trichromats and the peak sensitivities of their cones invariably lie near 430, 535 and 565 nm. This consistent pattern contrasts with the results reported earlier for New World monkeys and with the results reported here for Tupaia glis. The trichromacy of frugivorous catarrhine monkeys may have co-evolved with a particular class of coloured fruit. Short-wave cones were rare in all species. The ratio of the numbers of middle-wave and long-wave cones varied between individual animals, but had an overall value close to unity. In the case of all the species examined here, we have recorded a photostable pigment in the inner segments of rods and cones. The latter pigment has a peak sensitivity close to 420 nm and an absorbance spectrum that is narrower than that of a photosensitive visual pigment.

Animals↗
PubMed1993

Spectral sensitivities of the human cones.

Transient chromatic adaptation produced by an abrupt change of background color permits an easier and closer approach to cone isolation than does steady-state adaptation. Using this technique, we measured middle-wave-sensitive (M)-cone spectral sensitivities in 11 normals and 2 protanopes and long-wavelength-sensitive (L-) cone spectral sensitivities in 12 normals and 4 deuteranopes. Although there is great individual variation in the adapting intensity required for effective isolation, there is little variation in the shape of the M- and L-cone spectral-sensitivity functions across subjects. At middle and long wavelengths, our mean spectral sensitivities agree extremely well with dichromatic spectral sensitivities and with the M- and L-cone fundamentals of Smith and Pokorny [Vision Res. 15, 161 (1975)] and of Vos and Walraven [Vision Res. 11, 799 (1971)], both of which are based on the CIE (Judd-revised) 2 degrees color-matching functions (CMF's). But the agreement with the M-cone fundamentals of Estévez [Ph.D. dissertation, Amsterdam University (1979)] and of Vos et al. [Vision Res. 30, 936 (1990)], which are based on the Stiles-Burch 2 degrees CMF's, is poor. Using our spectral-sensitivity data, tritanopic color-matching data, and Stile's pi 3, we derive new sets of cone fundamentals. The consistency of the proposed fundamentals based on either the Stiles-Burch 2 degrees CMF's or the CIE 10 degrees large-field CMF's with each other, with protanopic and deuteranopic spectral sensitivities, with tritanopic color-matching data, and with short-wavelength-sensitive (S-) cone spectral-sensitivity data suggests that they are to be preferred over fundamentals based on the CIE 2 degrees CMF's.

Color Perception↗
PubMed2003

Using synchrotron-based FT-IR microspectroscopy to study erucamide migration in 50-micron-thick bilayer linear low-density polyethylene and polyolefin plastomer films.

The diffusion of additives in thick (approximately 500 microns) single layer and multilayer films has been characterized using FT-IR microspectroscopy. The objective of this research was to investigate additive migration and concentration profiles in coextruded multilayer films of industrially relevant thicknesses. In particular, the investigation focused on the migration of an erucamide slip agent in 50-micron-thick coextruded bilayer films of linear low-density polyethylene (LLDPE) and a polyolefin plastomer (POP). Erucamide concentration profiles were successfully mapped using synchrotron-based FT-IR microspectroscopy. The synchrotron radiation helped to achieve a higher spatial resolution for the thin films. Meticulous sample preparation was needed to map the thin film samples. Results with FT-IR microspectroscopy showed that the additive-concentration profiles were relatively uniform across the multilayer-film thickness irrespective of the intended initial additive distribution. For example, a bilayer planned for 1 wt % erucamide in an LLDPE layer and no erucamide in a POP layer showed significant additive migration into the POP layer at the extrusion rates used. FT-IR microspectroscopy results also showed that more erucamide migrated to the surface of a POP layer than an LLDPE layer. Attenuated total reflectance (ATR) FT-IR spectroscopy was used to confirm the time-dependent increase of erucamide surface concentration and that the increase was more pronounced at the surface of the POP layers.

Coated Materials, Biocompatible↗
PubMed2003

Application of multivariate curve resolution for analysis of FT-IR microspectroscopic images of in situ plant tissue.

The chemometric techniques of multivariate curve resolution (MCR) are aimed at extracting the spectra and concentrations of individual components present in mixtures using a minimum set of initial assumptions. We present results from the application of alternating least squares (ALS) based MCR to the analysis of hyperspectral images of in situ biological material. The spectra of individual pure components were mathematically extracted and then identified by searching the spectra against a commercial library. No prior information about the chemical composition of the material was used in the data analysis. The spectra recovered by ALS-MCR analysis of an FT-IR microspectroscopic image of an 8-micron-cornkernel section matched very well the spectra of the corn storage protein, zein, and starch. Through the application of MCR, we were able to show the presence of a second spectrally different protein, which could not be easily seen using univariate analysis. These results demonstrate the value of multivariate curve resolution techniques for the analysis of biological tissue. The value of principal components analysis (PCA) for hyperspectral image analysis is also discussed.

Algorithms↗
PubMed2003

FT-IR microspectroscopic study of shot formation in melt-blown webs.

We used Fourier transform infrared (FT-IR) microspectroscopy to investigate the chemical nature of fibers and defects called "shot" in melt-blown webs. Spectral differences were observed and evaluated in light of known thermal and oxidative degradation reactions for conditions comparable to those we used for melt blowing. Three different isotactic polypropylene polymers were evaluated in terms of the amount of shot produced and the amount of oxidative degradation exhibited by fibers and shot particles from each polymer. Little oxidative degradation was observed in fibers and the amount of degradation in fibers varied little for the three polymers we evaluated. Substantially more degradation was observed in shot particles, and the amount of degradation varied among the three polymers. Compared to polymer bound for fibers, we concluded that high temperature and mechanical shear in the extruder may introduce more chain scission in polymer bound for shot particles. Autoxidation reactions may occur after melt exits the die, and our data indicated that more oxidative degradation occurred in polymer that became shot particles than polymer that became fibers. The most favorable site for oxidation seemed to be tertiary rather than methylene hydrogen. Overall, the thermal history of polymer that becomes shot particles may be significantly different than the thermal history of polymer that becomes fibers, and this difference may have influenced shot formation.

Hot Temperature↗
PubMed2003

Fluorescence microspectroscopy technique for the study of intracellular protoporphyrin IX dynamics.

We describe a technique designed to monitor the fluorescence dynamics of photosensitizers used in photodynamic therapy (PDT) at micrometer-scale locations within individual formalin-fixed cells. The accumulation of protoporphyrin IX (PpIX) within keratinocytes and fibroblasts. following incubation with 5-aminolaevulinic acid (ALA), is shown to be dependent upon both incubation time and cell proliferation status. Also, the process of photobleaching within these cells is demonstrated via the depletion in PpIX fluorescence emission during exposure to 532 nm light. All spectra show a progressive reduction of the 634 nm PpIX peak, following a bi-exponential decay that is consistent with a singlet oxygen mediated process. The rate of photobleaching, when plotted as a function of light dose, increases with reduced incident laser power. The generation of the hydroxyaldehyde-chlorin photoproduct (photoprotoporphyrin), as monitored by the increase in fluorescence emission centered on 672 nm, is also greatest when the lowest laser power is applied. When light is delivered in two fractions, PpIX fluorescence recovers during the dark period and there is an increase in bleaching rate at the onset of the second exposure. These results are qualitatively consistent with measurements performed in vivo, which demonstrate that the photodynamic dose is dependent upon fluence rate and oxygen status.

Aminolevulinic Acid↗
PubMed2003

New opportunities in micro- and macro-attenuated total reflection infrared spectroscopic imaging: spatial resolution and sampling versatility.

New opportunities exist to obtain chemical images using attenuated total reflection infrared (ATR-IR) spectroscopy. This paper shows the feasibility of obtaining FT-IR images with a spatial resolution of at least 3-4 microm using a Ge ATR objective coupled with an infrared microscope. The improved spatial resolution compared to FT-IR images obtained by the transmission method is due to the high refractive index of the ATR crystal, which gives a high numerical aperture and hence, a higher spatial resolution. FT-IR imaging with a conventional diamond ATR accessory has been investigated. This is the first time that FT-IR imaging is reported using such a versatile accessory based on a diamond ATR crystal. These results showed that a spatial resolution up to 13 microm can be achieved without the use of infrared microscope objectives. One advantage of the diamond element is that it allows pressure to be applied and hence, good contact to be obtained over the whole field of view.

Crystallography↗
PubMed2003

Spectroscopic evaluation of the effect of a red microalgal polysaccharide on herpes-infected Vero cells.

The sulfated polysaccharide obtained from a species of red microalga has proved to be a potent antiviral agent against various members of the herpes family. In the present study, we used microscopic Fourier transform infrared spectroscopy (FT-IR) to investigate differences between normal cells, those infected with herpes viruses, and infected cells treated with red microalgal polysaccharide. FT-IR enables the characterization of cell or tissue pathology based on characteristic molecular vibrational spectra of the cells. The advantage of microscopic FT-IR spectroscopy over conventional FT-IR spectroscopy is that it facilitates inspection of restricted regions of cell cultures or tissue. Our results showed significant spectral differences at early stages of infection between infected and noninfected cells, and between infected cells treated with the polysaccharide and those not treated. In infected cells, there was an impressive decrease in sugar content and a considerable increase in phosphate levels in conjunction with the infection progress. Our results also proved that sugars penetrated and accumulated inside cells treated with the red microalgal polysaccharide. These could have been sugar fragments of low molecular weight present in the polysaccharide solution, despite purification by dialysis. Such sugar accumulation might be responsible for a breakdown in the internal steps of the viral replication cycle.

Animals↗
PubMed2003

Semiautomated depositor for infrared microspectrometry.

The measurement of minute samples is desirable in many areas of research and analysis, including biological, environmental, and forensic sciences. The use of manual solution direct deposition, combined with surface evaporation, is a very useful and convenient method for the transfer of many analytes to a spectroscopic sampling window. For Fourier transform infrared (FT-IR) spectrometric analysis, high quality attenuated total reflection (ATR) and transmission analyses are possible with suitable solution deposits. If the sample area is very small, placement of the analyte deposit on the active area of the sampling accessory must be done carefully. To achieve this, a novel direct deposition system has been developed. This system, a series of valves attached to a glass nebulizer, generates deposits that are reproducible, and placement of the deposits is precise. The valves allow the sample to be loaded, helium airflow to expel the sample, and the nebulizer to be cleaned after deposition. To help contain the sample to a small area once deposition has taken place, a vacuum line is attached to the nebulizer. This simple semiautomated deposition system allows for higher sensitivity and run-to-run reproducibility for minute sample analysis.

Equipment Design↗
PubMed2003

Analysis of depth profiling data obtained by confocal Raman microspectroscopy.

The nominal depth resolution achieved in confocal Raman microscopy is on the order of a few micrometers. Often, however, the depth resolution is decreased by light refraction at the sample surface. The problem can be avoided with the use of an immersion objective and index matching oils. Through this intervention the instrument point-spread function (PSF) can be assumed to be independent of the depth of focus in the sample, and spatially invariant depth profiles can be acquired. In this work the instrument PSF was determined by measuring a depth profile of a thick uniform sample and calculating the first derivative of the depth profile curve. The first-derivative method was also used to determine sample thickness. Convolution with the PSF makes it possible to simulate the behavior of the instrument with different sample functions. It is also possible to use the instrument PSF to deconvolve depth-profiling data. Deconvolution reduces the blurring effect of the instrument and increases the depth resolution. Deconvolution can also be used in analysis of the sample surface position and in layer structure analysis. In this paper we show how the convolution integral can be used with the immersion sampling technique to determine the PSF and how the sample thickness can be determined.

Algorithms↗