Fourier transform infrared bacteria identification with the use of a focal-plane-array detector and microarray printing.
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Bacteria↗
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Results for “Microspectrophotometry”
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Characterization of the diffusion of polyethylene glycol in Streptococcus mutans biofilms by Raman microspectroscopy.
We used Raman microspectroscopy to investigate in situ the spatial distribution of the biomass in Streptococcus mutans biofilms. We used the CH stretching band to probe the organic matter and the area of the OH stretching band as an internal intensity standard, the biofilms being highly hydrated. The size of the biofilm regions that were mapped was 300 x 300 microm. We also recorded, in the confocal mode, the z profiles describing the biomass distribution as a function of depth in the biofilms. In our growth conditions, the biofilm is described as an approximately 75 microm thick mat completely covering the surface and includes columnar clusters with a diameter of approximately 100 microm surrounded by voids filled with water. Raman mapping was also used to examine the diffusion of HOD and polyethylene glycol with a molar mass of 10,000 (PEG-10k) in the biofilms. This study establishes that HOD can diffuse practically everywhere in the biofilms but that the penetration of PEG-10k is limited. There is a correlation between the restricted penetration of the macromolecule and the biomass content in the different regions of the biofilms. The method presented here provides a convenient approach to determine the diffusion of molecules, including antibacterials, in bacterial biofilms.
Biofilms↗
Photoreceptor differentiation during retinal development, growth, and regeneration in a metamorphic vertebrate.
To test the hypothesis that growth and regeneration of the adult retina involves a mechanistic recapitulation of retinal development, the patterns of photoreceptor differentiation were investigated in the developing retina, as well as growing and regenerating adult retina, of a metamorphic vertebrate, the winter flounder. Only one opsin, of type RH2 (a "green" cone opsin), was expressed in premetamorphic (developing) retina, and a corresponding middle-wavelength visual pigment was observed. In premetamorphic retinas there was no evidence for any other cone opsins or pigments, rods, rod opsin expression, or rod visual pigment. In contrast, a rod opsin (RH1) and three cone opsins (SWS2, RH2, and LWS) were expressed in postmetamorphic (adult) retina, and these opsins were consistent with the observed repertoire of visual pigments. During postmetamorphic retinal growth and regeneration, cones were always produced before rods, but the different cone types were apparently produced simultaneously, suggesting that cone differentiation mechanisms might change after metamorphosis. The results support the hypothesis that photoreceptor differentiation during growth and regeneration of the adult retina involves a recapitulation of mechanisms that control the sequence of photoreceptor production during retinal development.
Amino Acid Sequence↗
Monitoring cell cycle distributions in MCF-7 cells using near-field photothermal microspectroscopy.
Microspectroscopic techniques such as Fourier transform infrared (FTIR) have played an important role in "fingerprinting" the biochemical composition of cellular components. Based on structure and function, complex biomolecules absorb energy in the mid-infrared (lambda = 2-20 microm) yielding characteristic vibrational infrared (IR) spectra. However, optical detection FTIR microspectroscopy may not be suitable for IR-absorbing sample materials. Photothermal microspectroscopy (PTMS) permits the direct measurement of heat generated as a result of sample material absorbing radiation. This approach generates true absorption spectra and is implemented by interfacing a scanning probe microscope and an FTIR spectrometer. Detection is performed using a near-field ultra-miniaturized temperature sensor. Employing PTMS, IR spectra of MCF-7 cells were examined in spectral regions (900-2000 cm(-1)) corresponding to proteins, DNA, RNA, glycoproteins, carbohydrates, lipids, and levels of protein phosphorylation. As a cell passes through the cell cycle, its nuclear material decondenses and condenses and this has led to ambiguity as to whether the intensity of such spectral regions may be associated with the G(1)-, S- or G(2)-phases of the cell cycle. Cultured cells were tracked over a time course known to correspond to marked alterations in cell-cycle distributions, as determined using flow cytometry. Experiments were carried out in the absence or presence of lindane, a pesticide known to induce G(1)-arrest in MCF-7 cells. Significant (P < 0.05) elevations in spectral intensities were associated with exponentially growing cell populations, predominantly in S-phase or G(2)-phase, compared to more quiescent populations predominantly in G(1)-phase. Increases in the absorption band at 970 cm(-1), associated with elevated protein phosphorylation, were observed in vibrational spectra of exponentially growing cell populations compared to those exhibiting a slowing in their growth kinetics. These results seem to suggest that intracellular bulk changes, associated with transit through the cell cycle, can be tracked using PTMS.
Analysis of Variance↗
Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing.
Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.
Animals↗
Noninvasive measurement of total serum bilirubin in a multiracial predischarge newborn population to assess the risk of severe hyperbilirubinemia.
BACKGROUND: Jaundice in near-term and term newborns is a frequent diagnosis that may prompt hospital readmission in the first postnatal week. Hyperbilirubinemia, when excessive, can lead to potentially irreversible bilirubin-induced neurotoxicity. Predischarge risk assessment (at 24-72 hours of age) for subsequent excessive hyperbilirubinemia is feasible by a laboratory-based assay of total serum bilirubin (TSB). Hypothesis. Noninvasive, transcutaneous, point-of-care measurement of transcutaneous bilirubin (TcB) predischarge by multiwavelength spectral analysis, using a portable BiliCheck device (SpectRx Inc, Norcross, GA), is clinically equivalent to measurement of TSB in a diverse, multiracial term and near-term newborn population and predictive of subsequent hyperbilirubinemia. METHODOLOGY: We evaluated a hand-held device that uses multiwavelength spectral reflectance analysis to measure TcB (BiliCheck). The study population (490 term and near-term newborns) was racially diverse (59.1% white, 29.5% black, 3.46% Hispanic, 4.48% Asian, and 3.46% other) and was evaluated at 2 separate institutions using multiple (11) devices. The postnatal age ranged from 12 to 98 hours and the ranges of birth weights and gestational ages were 2000 to 5665 g and 35 to 42 weeks, respectively. All transcutaneous evaluations were performed contemporaneously and paired with a heelstick TSB measurement. All TSB assays were performed by high performance liquid chromatography, as well as by diazo dichlorophenyldiazonium tetrafluoroborate techniques. RESULTS: TSB values ranged from .2 to 18.2 mg/dL (mean +/- standard deviation: 7.65 +/- 3.35 mg/dL). The overall correlation of TSB (by high performance liquid chromatography technique) to TcB (by BiliCheck devices) was linear and statistically significant (r =.91; r(2) =.83; TcB =.84; TSB = +.75; standard error of regression line = 1.38; P <.001; n = 490 infants; 1788 samples). Similar regression statistics were evident in subset populations categorized by race (white: r =.91 [n = 289 infants]; black: r =.91 [n = 145 infants]) as well as by gestation (term: r =. 91 [n = 1625 samples]; near-term: r =.89 [n = 163 samples]). Intradevice precision was determined to be.59 mg/dL (2-3 measurements per infant with 1 device; n = 210 infants; 510 samples in a separate subset). Interdevice evaluation of 11 devices determined the precision to be.68 mg/dL (2-4 devices used for measurements per patient). In 23 of 419 of the study population infants who were in the 24- to 72-hour age range, the predischarge TSB values designated them to be at high risk for subsequent excessive hyperbilirubinemia (above the 95th percentile track on the hour-specific bilirubin nomogram). For these infants, the paired BiliCheck TcB values were all above the 75th percentile track (negative predictive value = 100%; positive predictive value = 32. 86%; sensitivity = 100%; specificity = 88.1%; likelihood ratio = 8. 43). CONCLUSIONS: Our data demonstrate the accuracy and reproducibility of the predischarge BiliCheck measurements in term and near-term newborn infants of diverse races and ethnicities. Infants with predischarge BiliCheck values above the 75th percentile of hour-specific TSB values on the bilirubin nomogram may be considered to be at high risk for subsequent excessive hyperbilirubinemia. Further studies are needed to assess the efficacy of this technique in preterm infants, those undergoing phototherapy, and those with TSB values of >/=15 mg/dL (>/=256 micromol/L).
Analysis of Variance↗
Time-resolved microspectrofluorimetry and fluorescence lifetime imaging of hypericin in human retinal pigment epithelial cells.
Hypericin is the active ingredient of the off-the-shelf antidepressant St. John's Wort. It is an effective phototoxic agent and its systemic administration at therapeutic doses could induce particular damage in the eye due to continuous light exposure. Hypercin is strongly fluorescent and its fluorescence properties can be monitored to investigate noninvasively its localization and interactions. To this aim, time-resolved microspectrofluorimetry and fluorescence lifetime imaging were used to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by retinal pigment epithelium (RPE) cells treated with Hyp at concentrations in the micromolar range (0.5-10 microM). In the presence of hypericin, the emission peaks at 600-605 nm and the fluorescence decay is best fitted with three lifetimes (5.5-7 ns, 1.9-2.5 ns and <0.8 ns). Spectral and temporal differences were observed between high (> or =5 microM) and low hypericin concentrations. In particular, upon increasing concentration, the emission spectrum of the slow component broadens and its lifetime shortens. The latter change is observed also when high concentrations are reached locally, due to more efficient localization within the cell.
Anthracenes↗
Infrared microspectroscopic characteristics of radiation-induced apoptosis in human lymphocytes.
Infrared microspectroscopic characterization of radiation-induced apoptosis was used as a new analytical tool to study the kinetics of apoptosis in human peripheral blood lymphocytes at the molecular level. This vibrational technique, which has already been used to investigate biomolecules in normal and tumor cells, allows the simultaneous detection of the biochemical changes in the various subcellular compartments. Normal circulating lymphocytes from five healthy human donors were given a single dose of 6 Gy ((60)Co) and deposited on ZnS windows for infrared spectral acquisition 1, 2 and 4 days after irradiation. Apoptosis was assessed simultaneously by flow cytometry analysis of lymphocytes displaying annexin V-positive staining, and by detection of the DNA laddering that is characteristic of apoptosis. The flow cytometry study showed that about 80% of sham-irradiated lymphocytes were annexin V(neg)/PI(neg) at 1, 2 and 4 days. One day after irradiation, 46% of irradiated lymphocytes were annexin V(neg)/PI(neg), 48% were annexin V(pos)/PI(neg), 5% were annexin V(pos)/PI(pos), and 1% were annexin V(neg)/PI(pos). These mean percentages were respectively 31, 59, 9 and 1 at day 2 and 23, 36, 30, and 11 at day 4. Irradiated lymphocytes presented a DNA laddering pattern characteristic of apoptosis from day 1 after irradiation. In the infrared spectra of irradiated lymphocytes, qualitative and quantitative changes were observed from days 1 and 2, respectively. In the range of 960-1245 cm(-1) mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structures, in particular an increase in the amount of beta structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids at day 4 after irradiation. These results showed that DNA is probably the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. Our results are in agreement with biochemical and morphological data on radiation-induced apoptosis of normal human circulating lymphocytes, and they demonstrate that infrared microspectroscopy may be useful for assessing the process of apoptosis at the molecular level.
Adult↗
Utilization of dried sludge for making ceramsite.
Dried sludge as additive for making ceramsite is a new effective approach for disposal of sludge. In this study sewage sludge, water glass and clay were chosen as the components, the optimal ratio of the components and the most appropriate conditions were obtained. The functions of primary components in the sintering process, porosity formation mechanism and solid phase reaction also have been discussed. The optimized process parameters were shown as follows: the ratio of dried sludge/clay (wt%) was 33%, ratio of adherent /clay (wt%) was 15%, sintering temperature was 1000 degrees C, sintering time was 10 min. Bulk density was 582 kg m(-3), particle density was 1,033 kg m(-3), water absorption was 9.5%, porosity was 43.7%. SEM, EDS, XRD and XRF analyses were also carried out. The results indicate that dried sludge as raw material is a good way for making ceramsite. Biological Aerated Filters (BAFs) with filter media of Guangzhou ceramsite, Jiangxi ceramsite, activated carbon and ceramsite (obtained in test) were selected to treat municipal wastewater. The average removal efficiencies of ceramsite (obtained in test) for turbidity, COD, SCOD and NH3-N were about 96.4%, 76.2%, 59.6% and 82.3% respectively and were higher than those of other ceramsites.
Absorption↗
Spectral transmittance of retinal colored oil globules re-examined with microspectrophotometer.
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Humans↗
Recent advances in the imaging of programmed cell death.
A deficiency or an excess of programmed cell death (apoptosis) is an integral component of autoimmune disorders, organ and bone marrow transplant rejection, and cancer. A technique to image programmed cell death would be useful in the development of drugs to treat these and others diseases, and to monitor the effectiveness of therapy. The most widely studied agent for the in vivo study of apoptosis is radiolabeled annexin V, an endogenous protein labeled with technectium-99m, now undergoing clinical trials in both Europe and the United States. While annexin V has been studied extensively in humans the precise mechanism(s) of uptake of this agent in vivo is unclear and needs further study. Other agents are also underdevelopment including radiolabeled forms of Z-VAD.fmk, a potent inhibitor of the enzymatic cascade intimately associated with apoptosis. MR imaging techniques and tracers also hold promise as methods to monitor apoptotic cell death. In this article we will review these and other imaging technologies for the non-invasive imaging of cell death. The mechanism(s) and latest data on the conditions in which cellular stress and early apoptosis occur will also be discussed in detail including potential new strategies for the targeting and novel therapeutic interventions of tissues and organs undergoing stress or apoptosis when cell salvage is still possible.
Amino Acid Chloromethyl Ketones↗
Agonist-induced Ca2+ mobilization in cultured bovine and human corneal endothelial cells.
We investigated the possibility that cultured corneal endothelial cells express receptors that are coupled to the phosphoinositide cycle/intracellular Ca2+ signaling pathway. Agonist-stimulated changes in intracellular calcium ([Ca2+]i) in single bovine and human corneal endothelial cells (BCEC and HCEC, respectively) derived from confluent cultures were measured by microspectrofluorimetry using the Ca(2+)-sensitive probe, fura-2. Total inositol phosphates accumulated during a 30 min incubation in the presence or absence of agonists was determined in Li+ containing medium with cells pre-labelled for 48 hrs with 10 microCi/ml 3H-myoinositol. Histamine (HA), ADP and ATP induced a rapid increase in [Ca2+]i. Subsequently, [Ca2+]i decreased to either a stable, agonist-dependent sustained elevation, or fell back to baseline to begin oscillatory fluctuations. The initial rise in [Ca2+]i was insensitive to removal of extracellular calcium (Ca2+o), whereas the stable elevations in [Ca2+]i and the [Ca2+]i oscillations required Ca2+o. In contrast, bradykinin (BK) and endothelin-1 (ET-1) elicited an initial rise in [Ca2+]i that returned to prestimulatory levels within 2 min despite the continued presence of agonist. The Ca(2+)-mobilizing agonists carbachol, phenylephrine, adenosine and substance P were all ineffective in elevating [Ca2+]i. Histamine-induced Ca2+ mobilization was inhibited by the H1-receptor antagonist triprolidine, but triprolidine had no effect on either BK or ATP stimulation of Ca2+ mobilization. In BCEC, 100 microM HA significantly increased total inositol phosphate accumulation (18.8-fold over unstimulated controls) and was 90% inhibited by 0.5 microM triprolidine. BK and ATP also significantly increased formation of inositol phosphates in BCEC.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mitotic frequency determinations and microphotometric Feulgen dye measurements in root tips.
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Cell Division↗
Microspectrophotometric analysis of accumulation of the fluorones K-fluorescein, rose Bengal and phloxine red in living plant cells.
Spectrophotometric investigations of dye solutions in different media and of living stained cells from the upper epidermis of the scaleleaf of Allium cepa were carried out with the dyes K-fluorescein, rose Bengal and phloxine red to elucidate the mechanism of the accumulation of these dyes in the cytoplasm, the nucleus and the cell sap. Thin layer chromatography and paper electrophoresis indicate that the K-fluorescein used here contains no detectable contaminants. Besides the main component, rose Bengal contains two components in small quantities with Rf values of 0.64 and 0.57, plus three more components in traces. Besides the two main components (Rf values of 0.83 and 0.73), phloxine red also contains five more components in traces. Electrophoretic investigations reveal that in aqueous solution the fluorones rose Bengal and phloxine red from pH 2.0-11 show a migration toward the anode. K-fluorescein from pH 2.9-10.4 shows a migration toward the anode, but at pH 1.9 a migration toward the cathode. By shaking aqueous solutions of K-fluorescein, rose Bengal and phloxine red at different pH values with different organic solvents, the above used stainings show different spectral absorption curves according to the polarity of the solvent. The position of the absorption maxima and the shape of the absorption curves of these three anionic dyes lead to the conclusion that the staining of the living cytoplasm and nucleus is due to ion accumulation by means of the "ion trap mechanism" within the aqueous phase of the cytoplasm (cytosol) and the nucleus. Adsorption of dye particles in the protein phase of the cytoplasm cannot be excluded. There seems to be a fundamental difference in the vital staining of the protoplasm by anionic and cationic dyes, the latter apparently accumulating as neutral dye molecules in the lipid phase of the protoplasm. The concentration of the dyes used in the living cytoplasm (cytosol) is approximately 0.2-0.05%. During natural and artificial displacement of K-fluorescein from the cytoplasm to the vacuole, it appears that accumulation of the dye within the vacuole is performed through an ion trap mechanism in the form of bivalent ions. Along with natural displacement, it is possible that ion accumulation also occurs in metabolic products.
Allium↗
DNA cytometry of musculoskeletal tumors. A review.
In conclusion, all the benign soft tissue tumors analyzed so far have been been diploid, whereas the malignant entities include both diploid or aneuploid variants. Further, there is a relationship between ploidy level and histologic malignancy grade. The main value of DNA analysis seems to be that it provides objective support for histologic diagnosis as to benignity and malignancy grade. Occasionally, a discrepancy between ploidy and grade is encountered. Apart from the fact that aneuploidy precludes benignity, it may prove that ploidy level in malignancy is a better predictor of clinical course than histologic grade. However, this can only be established by analyzing each malignant entity separately, because the validity of DNA cytometry probably varies with histogenetic tumor type.
Bone Neoplasms↗
Intracellular calcium waves accompany neutrophil polarization, formylmethionylleucylphenylalanine stimulation, and phagocytosis: a high speed microscopy study.
Using high sensitivity fluorescence imaging with shutter speeds approximately 600,000 times faster than those of video frames, we have characterized Ca2+ waves within cells in exquisite detail to reveal Ca2+ signaling routes. Polarized neutrophils exhibited a counterclockwise rotating ryanodine-sensitive juxtamembrane Ca2+ wave during temporal calcium spikes. During stimulation with fMLP, a chemotactic factor, two Ca2+ waves traveling in opposite directions around the perimeter of the cell emanated from sites of stimulation (the clockwise wave is verapamil sensitive). Phagocytosed targets exhibit counterclockwise Ca2+ waves traveling about their periphery originating from the plasma membrane. This study: 1) outlines the technology to observe Ca2+ signaling circuitry within small living cells; 2) shows that extracellular spatial information in the form of a chemotactic factor gradient is transduced into intracellular chemical patterns, which provides fresh insights in signaling; 3) suggests that a line of communication exits between the cell surface and phagosomes; and 4) suggests that spatiotemporal Ca2+ patterns contribute to drug actions.
Animals↗
6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking during pregnancy.
Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme's intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.
Cell Separation↗
Multiprobe fluorescence imaging and microspectrofluorimetry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology.
The dichotomy of cellular transformation versus differentiation does not preclude the hypothesis of a unified underlying mechanism that can switch either way as a result of growth factors, cell-membrane receptors, secondary messengers, integrating switch kinases and/or nuclear receptors. Its study for biopharmaceutical and biotechnological applications requires a methodology capable of dealing with such pleiotropy. In the multiprobe-multiparameter approach, one must remain wary of cumulative toxic effects and misinterpretations. 'Smart' instrumentation does not mean 'smart' probes. It turns out that the cell's own endogenous probes, the fluorescent coenzymes, may be akin to 'smart' probes, open to study in situ of many-fold interrelated pathways in cell energetics and dynamics. Resolution at the micro- and even nano-compartment levels is not altogether impossible. Thus an innovative search in terms of what may be called 'intracellular reconnaissance with fluorescent probes and biopharmaceuticals' necessitates recourse to multiple tentative probings along the pleiotropic mechanisms as far in resolution as one can go. Among the characteristic findings using this approach are: (i) morphometric alterations in the mitochondria and melanosomes of melanoma cells treated with azelaic acid; (ii) deregulation of mitochondrial control and extramitochondrial metabolism in similarly treated cells; (iii) considerable acceleration of NAD(P) transient kinetics in atractylate-treated L sarcoma cells; (iv) alterations of mitochondria and Golgi in fusion-deficient myoblasts; (v) tentative recognition of beta-glucosidase deficiency in Gaucher disease cells by the use of fluorescent and fluorogenic lysosomal probes; and (vi) UVA-induced accumulation of Schiff bases (a kind of accelerated photo-aging) in yeast and kidney epithelial cells. Because these studies utilize probing at whatever points along the concerned pathways become accessible, at first glance they may look disconnected. What and where is the connecting thread, for instance, between studying melanoma metabolism, melanosome morphometry, hepatocyte organelle morphogenesis and transformation, myotube organelle morphogenesis and fusion-non-fusion, and lysosomal activity in gene-deficient cells? In the mapping of the regulatory and deregulatory mechanisms involved in the switching of differentiation or transformation, each of the above topics carries an information content towards resolution of the pleiotropic puzzle. The integration of such information with increasing resolution and access to intracellular microdomains may ultimately allow focus on the precise target, the switch from differentiation to transformation or vice versa.
Animals↗