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Studies of otoconial development in a "giant-crystal" strain of chicks using scanning electron microscopy, polarized light microscopy, and X-ray crystallography.

Otolith formation was studied in a mutant strain of low-fertility Delaware chicks which exhibit an otolithic defect. In all chicks of this strain, otoliths were present as a fused crystal mass which contained abnormally large (giant) otoconia. Studies of the formation of such otoliths during embryonic development revealed that from the very earliest stages the otoconia were much larger than normal, and in the saccular and utricular otoliths formed a fused mass. These results are interpreted as supporting a hypothesis of the de novo formation of giant otoconia in this giant-crystal strain as opposed to the recrystallization hypothesis proposed for other, dissimilar mutant mammals and birds which also produce giant otoconia.

Animals

Modified procedure for the quantitative estimation of pore volumes in carious dental enamel by polarizing microscopy.

The polarizing microscope was used to make two estimates of pore-volume fraction in carious enamel. Estimates from form birefringence were always much lower than estimates from the intrinsic birefringence of the enamel solid and were too low for carious enamel. The discrepancy cannot be explained except in part as being due to re-precipitation of mineral during lesion formation. The discrepancy may arise largely from the unjustified assumption that the whole tissue acts as a Wiener composite body. Form birefringence is probably produced only by a fraction of the tissue. A model which reconciles the two pore-volume estimates and enables calculation of the composite body fraction and of the volume fractions of solid within this fraction and in the tissue as a whole is described. It appears that polarizing microscopy will require the conjoint application of other techniques for quantitative estimation of pore-volume fractions.

Birefringence

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy.

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Antibodies, Monoclonal

The effect of speed of deoxygenation on the percentage of aligned hemoglobin in sickle cells. Application of differential polarization microscopy.

We have used differential polarization microscopy, which provides images of linear dichroism, to measure the percentage of aligned hemoglobin (Hb) in 1086 deoxygenated red blood cells from subjects with sickle cell anemia. The percentage was found to be only slightly dependent on the speed of deoxygenation, thus showing that the percentage of aligned Hb was thermodynamically controlled (as has been found previously for the percentage of polymerized Hb in vitro). A slight decrease in the percentage of aligned Hb due to increasing speed of deoxygenation is primarily due to the increase in the number of cells containing no detectable aligned Hb. This class of cells was also the most variable between the different subjects studied. We were able to identify two other groups of cells that contain different numbers of domains of aligned Hb and show that these groups contain statistically different percentages of aligned Hb. The differences between these classes of cells was shown to be primarily due to different numbers of initial nucleation sites within each cell. It appears that the presence of preformed nucleation sites within cells at ambient oxygen tensions results in the thermodynamic control of aligned Hb polymer.

Algorithms

Fibrillar bodies in leukemic cells revealed by polarization microscopy.

An optical polarizing microscope with a good coefficient of extinction permits the visualization of the cytoplasmic fibrillar body in living preparations and smears of leukemic cells (human leukemias and the L 5222 experimental leukemia). These inclusions are not visible by phase contrast microscopy nor in fixed and stained smears. The detection in living cells of fibrillar bodies makes it possible to study directly the conditions for their formation and their reaction to the effect of certain drugs.

Actin Cytoskeleton

[Application of polarized microscopy and analytic electron microscopy in pneumoconiotic pathologic examination].

38 cases of pneumoconiotic pathologic slides have been reexamined by polarized microscopy, among this, 3 cases (every 1 case of silicosis, asbestosis, cement dusts pneumoconiosis) have investigated by analytic electron microscopy. Authors presented, crystal silica and asbestos fiber may be showing under polarized light, and often associated with carbon dusts or fibrotic proliferation. So that, polarized microscopy may be as a preliminary detect method for silica and uncoated asbestos fiber from histologic slide, according to its dimension, color, lightness, morphologic feature and position were differential diagnosis from other positive substance under routine pathologic examination, further demonstration must to be reduced to ashes and re-observation under polarized microscopy. Also we selected 3 cases of silicosis, asbestosis, cement dusts pneumoconiosis specimen prepared to ultramicrotomic slides and examined by H-800 electron microscopy, when discovered some particles (such as crystal, non-crystal or fibrotic dusts) in cytoplasm of macrophages or interstitial space then used EDAX estimated. Its elements distribution were conformity with dusts (silica, asbestos, cement dusts).

Anthracosilicosis

[On applicabilities of polarization microscopy in diagnostic histopathology (author's transl)].

Apart from its importance in ultrastructure research polarization microscopy turns out to be a valuable additional method in the daily hitological diagnostics of pathologist. By means of some examples the applicabilities of the polarization microscopy in this field are demonstrated. After some physical bases and practical hints for the use of polarization microscopes some staining reactions leading to an optical anisotropic effect are reported. Morever, it is reffered to the demonstration and characterization of crystals in biological fluids and tisdues. In our opinion, simple polarization optics should belong to the optical equipment of each pathologist.

Aorta

Crystalluria determined by polarization microscopy. Technique and results in healthy control subjects and patients with idiopathic recurrent calcium urolithiasis classified in accordance with calciuria.

A retrospective study was done on the nature and degree of crystalluria in spontaneously voided fasting and postprandial urine of patients with recurrent idiopathic calcium urolithiasis (RCU) divided into normocalciuria (20 males, 20 females) and hypercalciuria patients (20 males, 20 females), and controls (20 males, 20 females). The crystals were obtained using a filter technique and identified by microscopy. In addition, individual data, clinical chemistry variables and indices reflecting the risk of calcium phosphate and calcium oxalate crystallization were evaluated. In contrast to findings of other investigators of crystalluria we observed only a few crystals on the filters. The most frequently occurring phases were (in this order) a urate-containing phase (tentatively termed uric), an amorphous calcium phosphate phase (tentatively termed isotropic) and a phase of spheroid-like particles, not yet definitely characterized (tentatively termed spheroid). Calcium oxalate crystals were found only exceptionally. There was no relationship between the degree of calciuria (normo- versus hypercalciuric RCU) and crystalluria. Among RCU, males generally had a predominance of the isotropic, females of the spheroid phase, as compared with controls. Also, RCU females were generally obese, and their spheroid score and lean body mass correlated negatively and significantly. The calcium phosphate and calcium oxalate risk indices were always low in normal individuals, higher in RCU. Patients of both sexes with urinary stones had normal parathyroid gland function, but higher total calcium in fasting serum and higher urinary pH as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen.

A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dyes is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5-6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.

Azo Compounds

[Spheno-occipital synchondrosis--a fluorescence and polarization microscopy study in Cercopithecus aethiops monkeys].

On 17 Cercopithecus aethiops monkeys we investigated with the method of the polychromic sequential dye marking system the histomorphology as well as the dynamics of growth and calcification of the spheno-occipital synchondrosis. The age of the animals ranged from change to teeth to late adolescence. The animals had been divided into four different groups: I: late change of teeth; II: young adult; III: fully grown; IV: late adolescence. In the first group the spheno-occipital synchondrosis showed no ossification in the gap which is filled with cartilage. It showed the characteristic structure with a central zone of equally distributed chondrocytes. Adjacent to this we found a zone of proliferation cell hypotrophy and cell degeneration. With increasing age there is decrease of the density of cells (groups II to IV) and after change to teeth the synchondrosis starts to ossify (group II). Due to the ossification the synchondrosis subdivides into different cartilage regions. We found that the closing of the synchondrosis started in the cranial region and progressed toward the caudal region. During this procedure the synchondrosis never ossified completely. Several cartilage regions persisted uncalcified until late adolescence. Interstitial growth of the synchondrosis was found until the end of the change of the teeth (group I). This growth which was always found in a sagittal direction ceased after bony connections had been formed between both poles of the synchondrosis. The sphenoidal and occipital pole of the synchondrosis showed equal growth potential.

Aging

Compensator transducer increases ease, accuracy, and rapidity of measuring changes in specimen birefringence with polarization microscopy.

An instrument has been designed to improve substantially the efficiency and convenience of measuring specimen birefringence retardation (BR) with a conventional Brace-Köhler compensator. The design is based on the precise transduction of the angular position of the compensator's Vernier dial to an easily visible red-illuminated display on a digital voltmeter. The instrument display is accurate to within 0.1 degrees over a range of +/-25 degrees in the compensator vernier dial position which is similar to the precision obtainable with an ordinary vernier and the Brace-Köhler method of measuring BR of biological specimens. For convenience in data reduction the display can also be read directly in nanometre units, with a similar precision for specimen BR in the range of 5 nm or less which is typical of birefringent fine structures in living cells.

Birefringence