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We estimated the long-range effects of air gun array noise on marine mammal communication ranges in the Southern Ocean. Air gun impulses are subject to significant distortion during propagation, potentially resulting in a quasi-continuous sound. Propagation modeling to estimate the received waveform was conducted. A leaky integrator was used as a hearing model to assess communication masking in three species due to intermittent/continuous air gun sounds. Air gun noise is most probably changing from impulse to continuous noise between 1,000 and 2,000 km from the source, leading to a reduced communication range for, e.g., blue and fin whales up to 2,000 km from the source.

The European Marine Strategy Framework Directive requires European member states to develop strategies for their marine waters leading to programs of measures that achieve or maintain good environmental status (GES) in all European seas by 2020. An essential step toward reaching GES is the establishment of monitoring programs, enabling the state of marine waters to be assessed on a regular basis. A register for impulsive noise-generating activities would enable assessment of their cumulative impacts on wide temporal and spatial scales; monitoring of ambient noise would provide essential insight into current levels and any trend in European waters.

Cognitive impairment in patients with bipolar disorder (BD) is not restricted to symptomatic phases. It is also present in euthymia. There is evidence of differences in the brain's structure between bipolar patients and healthy individuals, as well as changes over time in patients. Lithium constitutes the gold standard in long-term prophylactic treatment. Appropriate therapy that prevents new episodes improves the disease's course and reduces the frequency of harmful outcomes. Interestingly, preclinical data suggest that lithium has a (additional) neuroprotective effect. There is limited data on its related effects in humans and even less on its long-term application. In this multi-center cross-sectional study from the International Group for the Study of Lithium-treated Patients (IGSLi), we compared three groups: bipolar patients without long-term lithium treatment (non-Li group; <3 months cumulative lithium exposure, ≥24 months ago), bipolar patients with long-term lithium treatment (Li group, ongoing treatment ≥24 months), and healthy subjects (controls). Strict inclusion and exclusion criteria were defined; the inclusion criteria for patients were diagnosis of BD types I or II, duration of illness ≥10 years, ≥5 episodes in patient's history and a euthymic mood state. Neurocognitive functioning was assessed using the Wechsler Adult Intelligence Scale-Revised (WAIS-R), the California Verbal Learning Test (CVLT), and a visual backward masking (VBM) task. A total of 142 subjects were included, 31 in the non-Li and 58 in the Li group, as well as 53 healthy controls. Treated patients with long-standing BD and controls did not differ significantly in overall cognitive functioning and verbal learning, recall, and recognition; regardless of whether lithium had been part of the treatment. Patients, however, demonstrated poorer early visual information processing than healthy controls, with the lithium-treated patients performing worse than those without. Our data suggest that bipolar patients with a long illness history and effective prophylactic treatment do not reveal significantly impaired general cognitive functioning or verbal learning and memory. However, they are worse at processing early visual information. Accompanying volumetric and spectroscopic data suggest cell loss in patients not treated with lithium that may be counterbalanced by long-term lithium treatment.

A panoptic histological stain, PHTA, is introduced for routine use in developmental biology. The protocol is based on three dyes, hematoxylin and, after tannic acid treatment, phloxine B and azure B. It was optimized for differential staining of extracellular matrix, cytoplasm and chromatin. The method is quick, inexpensive and well-reproducible. The mouse placenta is used here to demonstrate the excellent suitability of PHTA for routine morphology.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.

The purpose of the present study was to investigate the influence of chromatin compactness on the kinetics of acid hydrolysis in the Feulgen reaction in cytology. Tissue imprints of rabbit liver, of human bronchial carcinoma and of human blood smears, fixed with alcohol, formaldehyde or with Böhm's solution with and without prior air drying, were stained with a standardized pararosanilin-Feulgen reagent. The time for hydrolysis varied between 7.5 and 120 min. The integrated optical density (IOD) of the cell nuclei was measured with an image analyzer (IBAS 2000). Cells with condensed chromatin (lymphocytes, small cell carcinoma, formaldehyde fixed cells) showed a slow increase of staining intensity and late plateau phase as compared with cells with decondensed chromatin. DNA in condensed nuclei was less susceptible to acid hydrolysis. The degree of chromatin compactness which determines the sensitivity of DNA to hydrolysis is influenced by the type of fixation, cell type and by the functional status of the cell. The conclusion is that Feulgen staining intensities of cells with different degrees of chromatin compactness cannot be compared unless measured in the respective plateau phases of the relevant hydrolysis curves which must be determined individually for each cell type.

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)

FAZYTAN, a system for fast automated cell segmentation, cell image analysis and extraction of nuclear features, was used to analyze cervical cell images variously stained by the conventional Papanicolaou stain, the new Papanicolaou stain and hematoxylin and thionin only; the last two dyes are used as the nuclear stains in the two versions of the Papanicolaou stain. Other dyes were also tried in cell classification experiments. All cell images in the variously stained samples could be described by the same nuclear features as had been adapted for the discrimination of conventional-Papanicolaou-stained cells. Variances were lower for thionin-stained cells as compared with hematoxylin-stained cells. By application of spectrophotometry, it was confirmed that the spectra of the cytoplasmic counterstains are superimposed on those of the nuclear stains. It appears that a variety of dyes are suitable as cytologic stains for cell classification by the FAZYTAN system, provided that they achieve sufficiently strong nuclear-cytoplasmic contrast by precisely delineating the chromatin texture.

The staining patterns of a modified Papanicolaou staining method and of its components are compared with those from the common Papanicolaou method. The staining results are investigated by the CIE-DIN-standardized color analysis. The medical investigator is able to discriminate more colors than this cytophotometric method, but this method is superior in the exact determination of colors. For neither of the both Papanicolaou staining methods it is possible to indicate one or two wavelengths for cytophotometric measurement using narrow-band filters which allows an optimal automated separation of nuclei, cytoplasms and background of all cell types. Keeping to this usual color analyzing method there is a better evaluation of the common Papanicolaou staining patterns. More saturated colors in the cyanophilic cytoplasms with the Papanicolaou staining patterns and in the eosinophilic cytoplasms with the investigated modified Papanicolaou staining patterns would be desirable. Since the resulting color patterns are similar from both stains, the modified Papanicolaou method has to prefer if it proves to be more reproducible.

In the present study we have investigated the uptake of cationic thiazine dyes and of the anionic Eosin Y by red blood cells (RBCs). Blood smears were stained with Azure B-Eosin Y, Methylene Blue-Eosin Y, Thionin-Eosin Y and with the cationic and anionic dyes alone at varying concentrations. Dye content of erythrocytes was measured with a Vickers M 85a microdensitometer. Nuclear chromatin features of white blood cells were investigated with the IBAS 2000 image analyser. Azure B favoured Eosin Y uptake of RBCs remarkably, and vice versa. There was no clear indication of that type of molecular interaction which characterizes the generation of the colour purple on polyanions. Methylene Blue and Thionin left Eosin Y uptake unaffected, but contamination of the standard Azure B-Eosin Y stain with Methylene Blue obviously affected Azure B-Eosin Y uptake, probably by competition of Methylene Blue and Azure B binding in RBCs. Furthermore, Methylene Blue contamination of the standard stain increased the rate of error in image analysis of white blood cell nuclei due to variations of staining intensity. For cytophotometry and image analysis of blood smears the standard Azure B-Eosin Y stain is by far superior to the commercial stain which normally is contaminated with Methylene Blue and some of the lower azures.

A simple and well reproducable modification of the Papanicolaou stain is reported. The most important modification is the replacement of the natural stain hematoxyline by the qualitively excellent synthetic stain thionin which is immediately added to the alcoholic fixation solution for a fixation stain. The high labor intense alternation between alcohol water and alcohol is thus eliminated. The quality of the nuclear staining is very good. The fixation staining with thionin without counter-staining of the cytoplasma may be used for a rapid thionin stain which gives sufficient cytoplasmatic staining by the thionin for a rapid cytological smear for cancer. The counter-stain of the cytoplasma was also simplified. The over-all staining of this simplification of the pap smear is comparable to that of the original papanicolaou stain. This modification of the papanicolaou stain fulfills requirements which a stain in competition with the original papanicolaou stain should have. 1. The modification is qualitively equal to the original method, 2. the staining method is simpler, 3. the staining method is well reproducable, 4. the staining method is more economical.

The morphologic and spectroscopic characteristics of a new and reproducible modification of the Papanicolaou stain are briefly described. The main features of this modification are (1) replacement of the natural dye hematoxylin by the synthetic and chemically well defined dye thionin, (2) introduction of an alcoholic counterstain consisting of eosin gamma and fast green FCF only and (3) employment of alcoholic solutions only. The absorption characteristics of hematoxylin and thionin bound to chromatin are influenced by the cytoplasmic counterstaining, especially by the two green dyes, the absorption peaks of which are close to those of the nuclear stains. The implications of these results for visual and automated cytologic diagnosis are briefly discussed.

A standardized thionin-eosinic acid stain was developed as a quick and highly reproducible staining method for bronchial cytology. Bronchial smears and paraffin-embedded sputum samples were stained with thionin-eosin and with the conventional hematoxylin-eosin Y. Spectral absorption characteristics and staining intensity of thionin-eosin-stained cells were investigated by means of cytophotometry. The staining pattern of thionin-eosin is very close to that of the hematoxylin-eosin stain; the contrast between nucleus and cytoplasm is significantly higher for thionin-eosin. Thionin-eosin can be used for "dye-fixation" of cytologic smears and tissue imprints. Blueing and differentiation (as for hematoxylin-eosin) is not required for thionin-eosin; thus, fixation and staining can be performed within two minutes. The spectral absorption characteristics of thionin-eosin allow reliable automated cytophotometric discrimination of cell nuclei and cytoplasm. The standardized thionin-eosin stain is recommended as a substitute for the hematoxylin and eosin stain in bronchial cytology.

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.

An extensive review is presented of the various factors involved in the standardization of dyes and stains for cytologic material, which is a crucial point in achieving objective and reproducible measurements of cells for recognition by automated microscopy (high-resolution cell image analysis; automated cell pattern recognition [ACPR]). Two principal methods of standardization are considered at length: (1) standardization based on the physical and chemical characterization of the dyes (physicochemical standardization), as adopted by the German Bureau of Standards, and (2) standardization based on direct visual assessment of stain performance (performance standardization), as adopted by the United States Biological Stain Commission. The procedures, achievements and problems of both methodologies are discussed, as are such related matters as staining time, solution pH, etc., especially with regard to the two most important stains in diagnostic cytology: the Papanicolaou stain and the Romanowsky-Giemsa stain. The standardization of these two stains is considered in detail, with an eye towards their suitability for ACPR. The mechanisms of the staining results produced by these stains are examined, as are the extant problems with each. The Papanicolaou stain, while neither standardized nor stoichiometric, has proven to be of use in cytophotometry. Similarly, the Romanowsky-Giemsa stain, while standardized, is also not a stoichiometric stain. Yet both have been successfully used in some aspects of ACPR; no "ideal" stain has yet been found that would make cell samples ideally suited for machine evaluation. It is concluded that the standardization of biologic dyes and stains, which can contribute to the success of ACPR, should be undertaken by multidisciplinary expert panels, in which the current concerned organizations could play a role. It is also concluded that ACPR may, in fact, contribute to the standardization of dyes and stains: that computer-directed morphometry and automated image analysis with sophisticated statistical analysis may become major tools in the validation of data on staining. For example, computer analysis could be used to pinpoint the particular variant of the Papanicolaou stain that leads to the best overall reproducibility of cell descriptors, in effect achieving "preliminary standardization by computer judgment of stain performance."

The advantages of the Commission Internationale de l'Eclairage (CIE) color measurements and the use of the standardizable purified azure B-eosin Y stain, as compared to the commonly used commercial Giemsa stain, were studied. Color measurement and analysis according to the CIE standards allows the comparison of color measurements from different slides stained by either the same or different procedures. Moreover, measurements from different cytophotometric systems can be compared. The results can also be directly correlated with human visual subjective color estimates and thus used to quantify the human observations. The color information in the air-dried cell specimens, which differs from the color characteristics of the components of the staining solution, was measured and analyzed.

Since cellular features can be visualized by proper staining of the biologic substrate, the interactions between the biologic dye and the substrate provide the parameters for automated cell recognition. A stock of 295 acridines and 19 derivatives of anthracene were tested as possible new compounds suitable as fluorescent probes in cytochemistry. Special attention was focused on marking fixed and unfixed cancer cells exfoliated from various organs. The results confirmed observations about the strong metachromatic properties of 3-aminoacridines and 9-aminoacridines. 9-amino-4-methyl-acridine showed an exceptionally high fluorescence of basophilic cellular structures. In addition to 4-aminoacridine, many quinacrine analogues, acridines lacking amino groups, 2-aminoacridine and some benzoacridines stained lipid droplets. A great variability in staining viable nuclei was found. 3-aminoacridine was superior as a Schiff's reagent, even in comparison to acriflavine. Not only aminoacridines but also pyrazol-anthracenes showed valuable properties as fluorochromes. Some further compounds (for instance, 9-aminoacridines) were accumulated in cytoplasmic vacuoles in a manner similar to that of the acridine orange. It is hoped that some of these compounds will prove useful in marking cancer cells.

The standardized Romanowsky-Giemsa-Stain, adapted for use in histology, is recommended as a suitable technique to assess effects of fixatives. The stain consists of two dyes only--Azure B and Eosin Y--but gives a polychrome and reproducible staining pattern. Most important is the colour purple which results from Azure B-Eosin Y molecular interaction. A collection of fixative effects on the RG-staining pattern is given. They are not easily revealed by other equally simple histochemical techniques. Of special interest is the fixative-dependent development of the colour purple on various biological substrates. For instance, both formaldehyde and acrolein allow the generation of this colour on collagen fibers but only after formaldehyde, hardly after acrolein, will the full colour purple appear on chromatin. A list of substrates, RNA among them, is presented which will not stain purple after any of the fixatives employed. The results are briefly discussed.

It had been observed that cell films where DNA had been partially digested by DNase stained differentially by the standard RG stain and a representative commercial Giemsa mixture. The reasons for this divergence were experimentally investigated. The lower concentration of Azure B in the commercial Giemsa mixture as compared with the standard RG stain emerged as the most likely factor causing the divergence of staining behaviour. A correlation of this assumption to the theory of the RG stain is tentatively suggested.

A new method is described for staining blood and bone marrow smears. It is characterized by the presence of only two dyes, purified azure B and eosin in methanol, as stock solutions. Staining results are equivalent to those obtained by using the traditional dye mixtures according to May and Grunwald, Giemsa, Leishman or Wright. Different from these azure B-eosin staining can be standardized and is easier to be handled. Correlations between the azure-B-eosin and May-Grunwald-Giemsa (MGG) staining methods are briefly discussed.

This paper reviews the nature of Romanowsky staining and the relationship between Romanowsky dyes and the Romanowsky-Giemsa effect (RGE). On blood and bone marrow smears the RGE is characterized by a purple colouration of nuclei and neutrophil granules. The nuclear purple contrasts strongly with the blue cytoplasmic staining of cells rich in RNA. Requirement for the occurrence of RGE are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used, but it can be replaced by the erythrosins. Full halogenation of the fluorescein (four atoms of bromine or iodine) is not necessary. Phloxine and rose bengal are unsuitable. 3 An appropriate substrate: These are proteins with acidic side groups or proteins bound to a polyanion. For the interaction with the dyes substrates must provide a suitable three-dimensional network which is why the RGE is not obtained in solutions. A tentative theory of RGE is advanced and briefly discussed.