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[Implementation of the immunological method for improvement on accurate platelet counts].

Rapid and accurate platelet counting is clinically required in severe thrombocytopenia. Prophylactic platelet transfusions are usually indicated in thrombocytopenia with platelet counts less than 20,000/microliter. It was recently reported that the confidence lower limit of platelet counts by automated blood cell counter is about 14,000/microliter. Clinical blood samples occasionally contain red-cell fragments or large platelets. In these cases, platelets should be counted by the phase-contrast microscopy. However, this manual operation is accurate but not precise and needs complicated technique. Abbott has developed an immunological platelet counting method by CELL-DYN 4000. We measured platelet counts in 137 blood samples from thrombocytopenic patients. These samples included red-cell fragmentation and large platelets on blood smears. We compared platelet counts with the immunological method(PLTimm) to those with Brecher-Cronkite, the optical(PLTo) and the impedance method(PLTi). PLTimm correlated more closely with the phase-contrast microscopy counts than PLTo or PLTi. In patients with microangiopathic hemolytic anemia, PLTo or PLTi could not exclude red-cell fragments, but PLTimm absolutely excluded red-cell fragments. In patients with giant platelets, PLTo or PLTi could not include large platelets but PLTimm included them and coincided well with platelet counts by the phase-contrast microscopy. These results indicate that the immunological method by CELL-DYN 4000 appears to be accurate and a very useful method for accurate platelet counts in severe thrombotybopenia.

Adult↗

Improved staining of extracellular polymer for electron microscopy: examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus.

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.

Azotobacter↗

Morphological observation on cell death and phagocytosis induced by ultraviolet irradiation in a cultured human lens epithelial cell line.

The purpose of this study is to observe dynamic morphological changes induced by ultraviolet (UV) irradiation in a cultured human lens epithelial cell line using electron microscopy, cell viability staining, time-lapsed videography and immunohistochemistry. Human lens epithelial cell line SRA 01-04 was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20% fetal bovine serum. Subconfluent cells were irradiated under a bank of UV lamps, which emitted 275-400 nm radiation with a maximum at 310 nm. The UV intensity was 20 microW cm(-2)at dosages from 0 to 10 mJ cm(-2). Alterations in the morphology of the living cells were monitored and recorded with phase-contrast microscopy and time-lapsed videography. At different times, the cells were fixed and examined by transmission electron microscopy (TEM), diamidinophenolindole (DAPI) staining, and in situ immunohistochemistry using TdT-mediated dUTP-biotin nick end labeling (TUNEL). Cell viability was also assessed with crystal violet staining. At low doses of UV exposure (2-5 mJ cm(-2)), time-lapsed videography revealed definitive cell death that appeared to be primarily apoptotic. The dead cell debris was engulfed and phagocytosed by neighboring living cells. Phase-contrast microscopy and TEM demonstrated that, at UV 10 mJ cm(-2), the cells not only showed typical apoptosis such as nuclear membrane shrinkage, chromatin condensation, and fragmentation into apoptotic bodies, but also necrosis such as swelling of the nucleus and cell body, and disruption of the plasma membrane. In support, DNA staining and in situ immunohistochemical reactions in the UV irradiated cells were both positive. The phagocytotic process was also seen with TEM. UV irradiation thus appears to cause both apoptosis and necrosis in the cultured human lens epithelial cell line. Active migration and phagocytosis of the cells appear to be stimulated by UV-induced damage. These findings may also aid in the understanding of UV injury and repair mechanisms of lens epithelial cells in vivo.

Apoptosis↗

Improvement of the micromethod for the limulus lysate test.

Frauch's micro-slide method was improved to facilitate the endpoint-determination of the Limulus test. Two precise observations, by inverted phase contrast microscopy and with a staining procedure, were newly performed as additions to the slide test. The staining procedure was proposed as an improved method for the Limulus test since it is simple and convenient. In the staining method, bromophenol blue (BPB) solution was used as the staining solution. A negative (-), a strong positive (++) and a weak positive reaction (+) were characterized by a "ring" formation, a "cloud-like" spread of gel and a "spot" in the "cloud" respectively. Since the distinction between (-) and (+) reactions was obvious in the proposed method, determination of the endpoint was easier than in the ordinary tube and Frauch's method. The sensitivity of the present method was equal to or higher than that of other methods. Inverted phase contrast microscopy was utilized to confirm the findings obtained by the staining method. The volume of the lysate used in this method was as little as 1/10 of that used in the tube method.

Blood Platelets↗

The use of urinary red cell morphology to determine the source of hematuria in children.

Urinary red cell morphology has been used to indicate the source of renal tract bleeding. A double blind study was undertaken to evaluate the reliability of urinary red cell morphology in predicting the source of hematuria in a pediatric population. Two independent observers used phase-contrast microscopy, one also using Nomarski differential interference microscopy, to examine 101 urine specimens from 90 pediatric patients with hematuria. 28 cases were subsequently excluded because the clinical diagnosis was uncertain. In the remaining 62 patients the laboratory diagnosis based on the erythrocyte morphology was compared with the primary clinical diagnosis. Observer 1 documented dysmorphic red cells in 40 of 42 patients with glomerulonephritis, and isomorphic red cells in 19 of the 20 cases of non-glomerular bleeding. Observer 2 documented dysmorphic red cells in 39 of the 42 glomerular lesions, and isomorphic red cells in all of the non-glomerular lesions. This represents a sensitivity for predicting glomerular hematuria of 95% for observer 1 and 93% for observer 2, and a specificity of 95% and 100% respectively. There was no definite advantage in the use of Nomarski compared to phase-contrast microscopy. These results confirm the accuracy of this technique in predicting the source of hematuria in pediatric patients.

Child↗

Differentiation of glomerular and non-glomerular hematuria in children by measurement of mean corpuscular volume of urinary red cells using a semi-automated cell counter.

Urine samples from 110 children and adolescents with micro- or macrohematuria were compared using phase-contrast microscopy and a semi-automated cell counter to differentiate glomerular from non-glomerular hematuria. Glomerular hematuria, defined by clinical criteria from biopsy and standard chemical evaluation, was observed in 73 patients (group 1): non-glomerular hematuria was found in 37 patients (group 2). The latter group underwent urological operation and had normal urine before operation. Mean corpuscular erythrocyte volume (MCVU) and percent of dysmorphic erythrocytes were determinated. To exclude the influence of mean erythrocyte volume of blood erythrocytes (MCVB), MCVB was determined and additionally the quotient of MCVU/MCVB was calculated (MCVUB). The percentage of dysmorphic erythrocytes differed significantly between the two groups ((75 +/- 13% in group 1 versus 38 +/- 27% in group 2 (mean +/- SD); p < 0.01), MCVU (34.0 +/- 11.1 fl in group 1 versus 55.5 +/- 16.3 fl in group 2; p < 0.01) and MCVUB (0.41 +/- 0.14 in group 1 versus 0.67 +/- 0.20 in group 2; p < 0.01). When glomerular hematuria was defined on the basis of more than 80% dysmorphic erythrocytes, the sensitivity of phase-contrast microscopy was 0.52, specificity versus 0.96 and efficiency 0.64. When glomerular hematuria was defined as < 50 fl MCVU, sensitivity was 0.92, specificity 0.57 and efficiency 0.80 and as < 0.06 MCVUB, sensitivity was 0.89, specificity 0.62 and efficiency 0.80. The correlation coefficient between MCVU and dysmorphic erythrocytes was -0.71 (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Associative phase separation of beta-lactoglobulin/pectin solutions: a kinetic study by small angle static light scattering.

Interpolymer complexation between beta-lactoglobulin (beta-lg) and pectin led to phase separation. Small angle static light scattering and phase contrast microscopy were used to monitor the phase separation of beta-lg/low-methoxyl or high-methoxyl-pectin (LM- or HM-pectin) dispersions as they were slowly acidified from pH 7 to 4 with glucono-delta-lactone (GDL). The monotonic decrease in scattered light intensity with the wave vector was associated with a nucleation and growth phase separation mechanism. Microscopic observations and turbidity measurements showed the increase of complex amounts with lower pH and at higher beta-lg/pectin ratios. The formation of intrapolymer complexes was initiated at pH 6.4 with the LM-pectin and at pH 5.0 with the HM-pectin. Local ordering with increasing amounts of small complexes was observed as scattered light intensity increased at intermediate q values. The beta-lg/LM-pectin complexes at the 5:1 and the 2:1 weight ratios and the beta-lg/HM-pectin complexes at 5:1 weight ratio have fractal structures. The formation of large amounts of small assemblies and sedimentation would be responsible for the decrease in the number and volume mean diameters and fractal dimension of beta-lg/LM-pectin complexes over time.

Kinetics↗

Analytical performance of the Iris iQ200 automated urine microscopy analyzer.

BACKGROUND: We evaluated the Iris iQ200 Automated Urine Microscopy Analyzer to find out if the instrument performed better than traditional visual bright field microscopy in detecting basic urine particles, as assessed against reference phase contrast microscopy. METHODS: The HUSLAB quality system was followed in planning and completing the evaluation process. The iQ200 instrument results from 167 mid-stream, uncentrifuged urine specimens were compared to those obtained with phase contrast reference microscopy, and to those with routine bright field microscopy. Linearity, carry-over and precision were tested according to well-established protocols. RESULTS: The iQ200 counted erythrocytes (RBC) at r=0.894 (R(2)=0.799) with Automated Particle Recognition (APR) software alone and at r=0.948 (R(2)=0.898) after re-classification. The performance for leukocytes (WBC) was r=0.885 with APR and r=0.978 after re-classification. The correlations of counting after user re-classification were r=0.927 for squamous epithelial cells (SQEP), r=0.856 for casts, and r=0.706 for non-squamous epithelial cells. The iQ200 showed good linearity and precision and no carry-over was detected. CONCLUSIONS: The Iris iQ200 was capable to count reliably RBC, WBC, and SQEP cells and to identify a fraction of bacteria and renal elements. Counting results equalled or exceeded that of routine bright field microscopy or earlier flow cytometric technology. The instrument eliminates manual sample preparation but requires a well-trained technologist for re-grouping of findings.

Automation↗

Morphological aspects in phase-contrast and transmission electron microscopy of senescent cultures of bovine aortic endothelial cells.

Some cultures of bovine aortic endothelial cells underwent an early senescence process, which may have been favoured by a special differential attachment trypsinization procedure. In order to better characterize this phenomenon, the cultures were observed at phase-contrast and electron microscopy at various passages. In phase-contrast microscopy, senescent cells were always larger than "younger" cells, and giant cells were very frequent. A great heterogeneity both of shape and size was present and the cells appeared shrunken, rigid and unable to cover the whole surface of the flask. In many senescent cells long fibres, running from one to the other side of the cytoplasm and often bridging over the nucleus were found. At transmission electron microscopy, together with a great abundance of rough endoplasmic reticulum, mitochondria and lysosomes, and the occasional presence of Weibel-Palade bodies, peripheric bundles of filaments, probably referable to actin or acto-myosin were found. These fibres may be interpreted as "stress fibres" and, as they are expression of a tension not accompanied by an effective cell motility, it seems of interest that they appear in senescent cells, which are often unable to cover the whole surface of flasks by migrating and subsequently proliferating in empty areas.

Animals↗

Morphology, development and behavior of Pneumocystis carinii observed by light-microscopy in nude mice.

The present paper describes the morphology, development and behavior of Pneumocystis carinii, especially of the trophozoites in the alveoli of conventionally raised BALB/c nude mice, by using phase-contrast microscopy, paraffin sections and semiultrathin sections embedded in JB-4 plastic. Under phase-contrast microscopy, trophozoites were ameboid in external appearance and 2 to 8 micron in diameter. Usually they have one or more lucid spherical vacuoles and one less lucid nucleus in the cytoplasm. Maturation and independence of intracystic bodies were observed in the developing cysts. The intracystic bodies were polymorphic, i.e. spherical, ameboid or elongated. The paraffin sections using the double staining of P. carinii with Gomori's methenamine silver nitrate (GMS) and Giemsa, showed a small number of mature cysts containing intracystic bodies and a large number of trophozoites within the characteristic honeycombed material in the alveolar spaces. In order to investigate the morphology and parasitizing behavior of the trophozoites and cysts as well as the response of alveolar tissue in more detail, semiultrathin sections of 0.5-2 micron thickness were made from the materials embedded in JB-4 plastic and stained with Giemsa. In lightly infected alveoli, some trophozoites and cysts were found to be closely attached to the Type I alveolar epithelial cells. In heavily infected alveoli, almost all alveoli were filled with trophozoites, cysts, debris of the host cells, and occasionally phagocytic cells (macrophages and neutrophils) containing cysts or trophozoites were found. It was noted that the cysts were very few in number (1%) compared with the number of the trophozoites in nude mice in a 0.4 mm2 area of 2 micron thick lung sections. The host tissue of nude mice in this study was not as strongly affected by the organisms as that in cortisone-treated rats and P. carinii pneumonia patients. In the present study the morphology of P. carinii found in nude mice was not different from that found in rats and in man.

Animals↗

LOCAL REDUCTION OF SPINDLE FIBER BIREFRINGENCE IN LIVING NEPHROTOMA SUTURALIS (LOEW) SPERMATOCYTES INDUCED BY ULTRAVIOLET MICROBEAM IRRADIATION.

Irradiation of the mitotic spindle in living Nephrotoma suturalis (Loew) spermatocytes with an ultraviolet microbeam of controlled dose produced a localized area of reduced birefringence in the spindle fibers. The birefringence was reduced only at the site irradiated, and only on the spindle fibers irradiated. Areas of reduced birefringence, whether produced during metaphase or during anaphase, immediately began to move toward the pole in the direction of the chromosomal fiber, even though the associated chromosomes did not necessarily move poleward. Both the poleward and the chromosomal sides of the area of reduced birefringence on each chromosomal fiber moved poleward with about the same, constant, velocity. On the average, the areas of reduced birefringence moved poleward with about the same velocities as did the chromosomes during anaphase. The area of reduced birefringence was interpreted as a region in which most, though not necessarily all, of the previously oriented material was disoriented by the irradiation. The poleward movement of the areas of reduced birefringence indicates that the spindle fibers are not static, nonchangeable structures. The poleward movement possibly represents the manner in which the birefringent spindle fibers normally become organized. All the experiments reported were on primary spermatocytes which completed the second meiotic division subsequent to the experimentation. Since both the irradiated and the control cells completed the two meiotic divisions, the movement and irradiation effects studied in the first division were nondegenerative.

Anaphase↗

Compensation of the inherent wave front curvature in digital holographic coherent microscopy for quantitative phase-contrast imaging.

An approach is proposed for removing the wavefront curvature introduced by the microscope imaging objective in digital holography, which otherwise hinders the phase contrast imaging at reconstruction planes. The unwanted curvature is compensated by evaluating a correcting wave front at the hologram plane with no need for knowledge of the optial parameters, focal length of the imaging lens, or distances in the setup. Most importantly it is shown that a correction effect can be obtained at all reconstruction planes. Three different methods have been applied to evaluate the correction wave front and the methods are discussed in detail. The proposed approach is demonstrated by applying digital holography as a method of coherent microscopy for imaging amplitude and phase contrast of microstructures.

Journal Article↗

Linear phase imaging using differential interference contrast microscopy.

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier-space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.

Fourier Analysis↗

Phase detection in acoustic microscopy.

The scanning laser acoustic microscope provides a relatively simple means by which speed of sound data can be collected from biological specimens. By employing a phase detection circuit, phase-contour lines can be superimposed on acoustic micrographs and digitized for direct speed of sound calculations. Because of the existence of mild field non-uniformities, the phase reference must be mathematically modeled. This is accomplished by employing a polynomial function and simple linear regression. Scattering can be studied by using a frequency-selective "dark field" approach. Speed of sound maps are readily produced from the phase data by applying the simple geometric relationships of wave propagation; however the theoretical limitations of the technique must be kept in mind. Using this approach, useful data have been obtained.

Acoustics↗

[Assessment of surface structure of blood cells by phase-contrast and scanning electron microscopy in children].

Study of red cell surface structure is a highly informative method for the assessment of the body status in health and disease. The authors have examined the surface red cell structure of infants in health and disease by phase-contrast and scanning electron microscopy and came to the conclusion on the high reliability of the data obtained by phase-contrast microscopy. They recommend this unsophisticated and informative method for clinical practice.

Acute Disease↗

Morphological and immunocytochemical characterization of osteoblast cultures from long bones of neonatal rats.

The present study reports a successful culture system of rat bone cells obtained from the bare long bone fragments of neonatal Wistar rats. The morphology, histochemical and immunocytochemical properties of the cultured cells were characterized by phase contrast microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), histochemical staining with alkaline phosphatase (ALP), and immunocytochemical staining with the anti-osteocalcin antibodies. Under phase contrast microscopy, the cultured rat bone cells changed their morphology from being slender with relatively-short cytoplasmic processes to becoming polygonal with longer processes, and subsequently flattened. These cultured bone cells formed bone nodules after 3 weeks. Under SEM, the cultured bone cell appeared polygonal with some microvilli. The TEM showed that their cytoplasm contained abundant endoplasmic reticulum and well-developed Golgi apparatus. Positive histochemical staining of ALP was also detected as a blue coloration and granular appearance in the cultured bone cells. Immunocytochemistry with the polyclonal and monoclonal anti-osteocalcin antibodies showed the localization of osteocalcin in both the cytoplasm and nucleus. All these data lead us to consider that the cultured bone cells in our system were probably osteoblasts from growing bone tissue in vitro. This provides a convenient test system for further studies on the regulation of the growth and metabolism of rat bone cells.

Alkaline Phosphatase↗

Fluorescence microscopy of phospholipid monolayer phase transitions.

Over many years, a detailed picture of the phase transitions in phospholipid monolayers at the air-water interface has been constructed from extensive studies of the force-area, viscoelastic and surface potential properties of phospholipid monolayers, yet the microscopic nature of the transitions has remained obscure. Recent investigations have focused specifically on these aspects. Through the use of fluorescence microscopy, electron diffraction and X-ray scattering experiments, in combination with data obtained by classical methods, a wealth of new information regarding the properties of monolayers undergoing phase transitions has been generated. Direct observation of fluid-solid phase coexistence at the air-water interface has been achieved with fluorescence microscopy and on solid supports with electron microscopy. The fluid-solid coexistence region has been studied most thoroughly to date, but regions of gas-fluid and fluid-fluid phase coexistence have also been detected. Numerous factors govern the properties of the coexistence region: however, the prominent features can be explained in terms of a competition between forces: long-range electrostatic forces and short-range attractive forces. In this review these recent experimental findings and theoretical interpretations are summarized.

Electrochemistry↗