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Results for “Microspectrophotometry”

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At least 19 recordsLinked to original sources

PubMed1996

Evaluation of ploidy of mature canine megakaryocytes, using Feulgen staining and microspectrophotometry.

OBJECTIVE: To evaluate megakaryocyte size and ploidy, using Feulgen staining and microspectrophotometry, in adult dogs with normal platelet count. ANIMALS: Group A contained 8 and group B contained 11 adult dogs. PROCEDURE: Megakaryocytes were evaluated by light microscopy and staged according to maturation status. Stage-III megakaryocytes were measured and mapped for future relocation. Bone marrow aspirates were destained and restained, using the Feulgen method. Previously identified stage-III megakaryocytes were measured for DNA content, using microspectrophotometry. RESULTS: Megakaryocyte size correlated with ploidy values, and mean sizes within ploidy groups were significantly (P < 0.05) different from each other for both groups. The model ploidy value of stage-III megakaryocytes, which represented 18% of the total megakaryocyte population of the combined groups, was 32N. This is in contrast to results of flow cytometric studies, which indicated that the modal ploidy value for all canine megakaryocytes was 16N. CONCLUSIONS: Reasons for the disparate results between microspectrophotometric techniques and flow cytometry include maturation stage of the megakaryocyte population evaluated and percentage of megakaryocytes within that maturation stage. Flow cytometric methods, which evaluate all megakaryocytes detectable by antibody, may include cells still capable of DNA synthesis, resulting in a shift in the observed modal ploidy value. Recognition of the difference between canine and human megakaryocyte ploidy distribution is important, particularly in studies in which the dog is used as an animal model for human megakaryocytopoiesis.

Analysis of Variance↗

PubMed1985

Monitoring of filamentous fungal growth by in situ microspectrophotometry, fragmented mycelium absorbance density, and 14C incorporation: alternatives to mycelial dry weight.

Monitoring of filamentous fungal growth by spectrophotometry is generally considered not feasible. This report describes the monitoring of growth of the filamentous fungi Trichophyton mentagrophytes, Rhizopus oryzae, and Sporothrix schenckii in broth by two new spectrophotometric methods and by 14C incorporation from [U-14C]glucose. Microcultures (200 microliter) were prepared in 96-well, flat-bottom microtiter trays, and macrocultures (4 ml) were prepared in glass vials proportionally scaled up from microcultures. Mycelium accumulation in microcultures was measured without terminating the cultures by in situ microspectrophotometry. Accumulation in macrocultures was monitored by uniformly fragmenting the mycelium with a Broeck tissue grinder and by measuring absorbance density in plastic cuvettes with a dual-beam spectrophotometer. Absorbance measurements were found to increase linearly with mycelial weight. In situ absorbance correlated with absorbance density of fragmented mycelium, indicating that both methods monitored growth equivalently. Both defined lag-, exponential-, and stationary-growth phases. Increases in 14C incorporation, absorbance, and mycelial dry weight were kinetically identical for macrocultures and microcultures of T. mentagrophytes. For R. oryzae and S. schenckii, with the exception of R. oryzae growing in microcultures, incorporation of 14C also defined lag, exponential, and stationary growth after selection of the appropriate isotope-specific activity. This incorporation correlated directly with absorbance. We conclude that in situ microspectrophotometry, fragmented mycelium absorbance density, and, to a lesser extent, 14C incorporation can be used to effectively monitor filamentous fungal growth.

Aspergillus niger↗

PubMed1979

Application of microspectrophotometry to biomedical research.

During recent years, the utilization of microspectrophotometry in biomedical research has increased at a rapid pace. This growth has been facilitated by both the advent of commercially available instruments which are reliable and manageable as well as improvements in histochemical techniques. This paper discusses the fundamental principles of visible light microspectrophotometry and important design criteria as well as briefly reviewing a wide range of applications. Special emphasis will be given to the Vickers M85 scanning integrating microspectrophotometer which is being routinely employed for both clinical and laboratory studies by a sizeable number of investigators.

Cell Nucleus↗

PubMed1998

Abnormal accumulation of porphyrin derivatives in the kidneys of Long-Evans Cinnamon rats, as evidenced by microspectrophotometry.

In the study described here we have revealed an abnormal accumulation of porphyrin derivatives in the kidneys of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. In addition, we have confirmed that the derivatives emitted red-orange light in renal sections under UV excitation. This renal red-orange emission has previously been identified as luminescence from cuprous metallothioneins [Cu(I)-MTs], which also accumulate in both the kidneys and liver of LEC rats. In this study, we measured the emission spectra of the luminescence in the kidneys using microspectrophotometry. The spectra of the renal red-orange emission resembled those of porphyrin derivatives rather than those of Cu(I)-MTs. We then extracted these derivatives from the kidneys. An abundance of porphyrin derivatives was established. A significant increase in the levels of the derivatives in the liver and urine of the LEC rats was also confirmed. These results provide evidence of a heme-metabolism abnormality in LEC rats.

Animals↗

PubMed1998

Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry.

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.

Alanine↗

PubMed1996

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry.

Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue, N,N'-Diethylpseudoisocyanine, and a modified PAS-method, and staining methods with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 microns-thick paraffin and 1 microns-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 microns apart using a Leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage. Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r = 0.900-0.995) with the fixed charge density curves from the adjacent cartilage. Also, the stain distribution was consistently uniform across the sections. In 1 microns-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.

Animals↗

PubMed2000

Visual pigments of African cichlid fishes: evidence for ultraviolet vision from microspectrophotometry and DNA sequences.

We have found evidence for ultraviolet visual capabilities in a Lake Malawi cichlid fish, Metriaclima zebra. Microspectrophotometry of single cones revealed a visual pigment with peak sensitivity at 368+/-4 nm. M. zebra also expresses a putative ultraviolet opsin gene whose sequence is closely related to the SWS-1 opsin for other fishes. Several other African cichlids have a functional copy of this UV gene in their genomic DNA, but do not appear to express this gene as adults. These results suggest that ultraviolet vision is important for some cichlid fishes. UV wavelengths should therefore be included in future studies of cichlid vision, behavior and color patterns.

Amino Acid Sequence↗

PubMedDate not supplied

Receptor demise from alteration of glycosylation site in Drosophila opsin: electrophysiology, microspectrophotometry, and electron microscopy.

In the delta Asn20 Drosophila stock, the N-linked glycosylation site of opsin in R1-6 receptors (Rh1) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rh1. MSP revealed minimal photopigment in delta Asn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for delta Asn20 and w9. Delta Asn20's R1-6 function is decreased 100-fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller R1-6 rhabdomeres in delta Asn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE(ol17), a deletion of the Rh1 gene, also has small rhabdomeres. Delta Asn20 and ninaE(ol17) lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rh1's coding sequence. Delta Asn20 and ora have surplus membrane while ninaE(ol17) does not. Freeze fracture reveals that delta Asn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, delta Asn20 interferes with either synthesis, deployment, or maintenance of opsin.

Animals↗

PubMed1988

Comparison between microspectrophotometry and cytofluorometry in measurements of nuclear DNA in human hepatocellular carcinomas.

A cell nuclear deoxyribonucleic acid (DNA) content analysis of paraffin-embedded materials of hepatocellular carcinoma in 18 patients was performed by two different methods, two-wavelength microspectrophotometry (paraffin section) and cytofluorometry (cell isolation). The DNA pattern was grouped into two types, low ploidy or high ploidy, and results achieved by each method were compared. The coincidence rate was 88.9% (16 in 18). Among the 14 with no or slight pleomorphism the coincidence rate was 100%, while it was only 50% (two in four) in those with moderate or marked nuclear pleomorphism. Therefore, when measuring the nuclear DNA content of cancer cells, the possibility of considerable differences between results of absorption and fluorescence cytophotometry in cases of a moderate or marked pleomorphism has to be considered.

Adult↗

PubMed1982

Studies on DNA content of hepatocytes in cirrhosis and hepatoma by means of microspectrophotometry and radioautography.

In order to clarify the underlying mechanism of malignant transformation from cirrhosis to hepatoma the cell kinetics of hepatocytes were studied in these two conditions. The content and synthesis of DNA in hepatocyte nuclei were investigated, by means of Feulgen-microspectrophotometry and tritiated thymidine radioautography, in cirrhotic and noncancerous parts of hepatoma with concomitant cirrhosis. The distribution of ploidy patterns was widely spread, from hypodiploid to hyperpolyploid, in the noncancerous parts of a cirrhotic liver containing hepatoma. In normal liver, each paired nuclear DNA content of a binucleate cell recorded almost the same amount, whereas in the noncancerous as well as in hepatoma cells much difference of DNA content was observed between the paired nuclei of the binucleate cells. The ploidy pattern of hepatocytes in patients with liver cirrhosis, who had developed hepatoma during follow-up periods of several months to several years, appeared to resemble that in noncancerous parts of hepatoma cases. On the other hand, the incorporation of tritiated thymidine into hepatocytes was found to be markedly increased in noncancerous parts as well as in cirrhotic liver developing hepatoma during follow-up periods. These results suggest the possibility that the hepatocytes in noncancerous parts of hepatoma have deranged cell-kinetics which might be a driving factor for the development of malignancy.

Acute Disease↗

PubMed1976

A comparison of two methods of preparing cells or nuclei for high-resolution microspectrophotometry.

Suspensions of isolated mouse thymus cells were subjected to two preparative methods: either they were dropped through several mm of 9:1 v/v ethanol-acetic acid fixative, allowed to stand for 1hr and then processed for staining; or they were fixed, passed through a graded ethanol series to 70% ethanol, centrifuged on to slides in a modified Shandon cytocentrifuge and then carried wet into the staining procedure. All preparations were stained by the Feulgen reaction and evaluated by high-resolution microspectrophotometry. While the two preparative procedures yielded similar results, there appeared to be less variability in the data obtained from the centrifuged cell populations.

Animals↗

PubMed1989

Demonstration of functional acetylcholinesterase on the soma of individual neurones of Aplysia by in vivo microspectrophotometry.

The presence of functional acetylcholinesterase is demonstrated in vivo on somatic membranes of single ganglionic neurones of Aplysia using concurrently microspectrophotometry and electrophysiology. The similarity of the effects of an irreversible blocker of acetylcholinesterase and of phospholipase C from Bacillus cereus suggests that acetylcholinesterase is anchored in the membrane via phosphatidylinositol.

Acetylcholinesterase↗

PubMed1976

The automation of microspectrophotometry of tissue sections.

A system for quantitative microspectrophotometry with automatic data acquisition is described. It is relatively simple and less expensive than presently-available equipment. Fitted with the appropriate software, the system permits various types of probe selection, with both selection and measurement made under individual optimal conditions.

Computers↗

PubMed1992

Visual receptor cycle in normal and period mutant Drosophila: microspectrophotometry, electrophysiology, and ultrastructural morphometry.

Visual pigment, sensitivity, and rhabdomere size were measured throughout a 12-h light/12-h dark cycle in Drosophila. Visual pigment and sensitivity were measured during subsequent constant darkness [dark/dark (D/D)]. MSP (microspectrophotometry) and the ERG (electroretinogram) revealed a cycling of visual pigment and sensitivity, respectively. A visual pigment decrease of 40% was noted at 4 h after light onset that recovered 2-4 h later in white-eyed (otherwise wild-type, w per+) flies. The ERG sensitivity [in w per+ flies in light/dark (L/D)] decreased by 75% at 4 h after light onset, more than expected if mediated by visual pigment (MSP) changes alone. ERG sensitivity begins decreasing 8 h before light onset while decreases in visual pigment begin 2 h after light onset. These cycles continue in constant darkness (D/D), suggesting a circadian rhythm. White-eyed period (per) mutants show similar cycles of visual pigment level and sensitivity in L/D; per's alterations, if any on the D/D cycles were subtle. The cross-sectional areas of rhabdomeres in w per+ were measured using electron micrographic (EM) morphometry. Area changed little through the L/D cycle.

Animals↗

PubMed1969

Microspectrophotometry and the photoreceptor of Phycomyces I.

By applying microspectrophotometry to the sporangiophore of Phycomyces blakesleeanus wild-type and the albino car-10(-) type II, absorption spectra were obtained for 1- to 5-day cultures. Spectra in the growing-zone of the wild-type during Stage IVb, taken from 0.1 to 3 mm below the base of the sporangium, show two distinctly different spectra: one is more characteristic of a carotene, the other of a flavin. Combined, these absorption spectra reproduce closely the action spectrum. For the albino car-10(-), which is deficient in carotenes, only the spectrum characteristic of lumichrome or a reduced flavin was found. A c-type cytochrome was isolated from both strains which, if coupled with a flavin, could permit a photoreversible oxidation-reduction system. Birefringent crystals were observed to be aligned in the growing zone in which the photoreceptor is believed to lie. Micro-spectrophotometry of these crystals shows absorption peaks similar to those of riboflavin crystals.

Birefringence↗

PubMed1993

Visual pigment bleaching in isolated salamander retinal cones. Microspectrophotometry and light adaptation.

Visual pigment bleaching desensitizes rod photoreceptors greatly in excess of that due to loss of quantum catch. Whether this phenomenon also occurs in cone photoreceptors was investigated for isolated salamander red-sensitive cones. In parallel experiments, (a) visual pigment depletion by steps of bleaching light was measured by microspectrophotometry, and (b) flash sensitivity was measured by recording light-sensitive membrane current. In isolated cones, visual pigment bleaching permanently reduced flash sensitivity significantly below that due to the reduction in quantum catch, and there was little spontaneous recovery of visual pigment. The "extra" desensitization due to bleaching was most prominent up to bleaches of approximately 80% visual pigment and reached a level approximately 1 log unit beyond that due to loss of quantum catch. At higher bleaches, the effect of loss of quantum catch became more important. Bleaching did not greatly reduce the maximum light-suppressible membrane current. A 99% reduction of the visual pigment permanently reduced the circulating current by only 30%. Visual pigment bleaching speeded up the kinetics of dim flash responses. All electrical effects of bleaching were reversed on exposure to 11-cis retinal, which probably caused visual pigment regeneration. Light adaptation in photopic vision is known to involve significant visual pigment depletion. The present results indicate that cones operate with a maintained circulating current even after a large pigment depletion. It is shown how Weber/Fechner behavior may still be observed in photopic vision when the contributions of bleaching to adaptation are included.

Absorptiometry, Photon↗

PubMed1975

Microspectrophotometry of rhodopsin and metarhodopsin in the moth Galleria.

Fresh, frozen sections of the photoreceptor layer of the compound eye of the moth Galleria have been examined by microspectrophotometry, using 4 times 8 mum measuring beams that sampled from approximately two to four rhabdoms. The principal visual pigment absorbs maximally at 510 nm (P510), and on irradiation is converted to a thermally stable, pH-insensitive metarhodopsin with lambda max at 484 nm (M484) and a 43% increase in molar extinction coefficient. Subsequently, short wavelength irradiation of the metarhodopsin photoregenerates some P510, but the absence of an isosbestic point the cycle of spectral changes is consistent with the presence of smaller amounts of violet-or ultraviolet-sensitive visual pigment(s) that also are converted to a blue-absorbing metarhodopsin. Difference spectra for both P510 and M484 were measured, using hydroxylamine. The 484-nm metarhodopsin is reversibly converted to a form with lambda max at 363 nm by high concentrations of glycerol. Dark regeneration of rhodopsin in vivo after several minutes exposure of thoroughly dark-adapted animals to full sunlight requires several days.

Animals↗

PubMed1978

Correction of modal DNA values obtained by microspectrophotometry and tests for their shifts.

The technical problems in the application of statistical analyses of the nuclear DNA contents measured by plug or two-wavelength Feulgen microspectrophotometry were discussed. The skewness of the nuclear DNA content unimodally distributed in a cluster could be corrected by logarithmic transformation. A mean value of the logarithmically transformed DNA contents was a more logical characterization of cancer than a graphically obtained mode of nontransformed values. Equations were derived to estimate the number of cells in the G1- and S-phases of mitotic cycles contained within the cluster and to estimate the correction factor of the mean of transformed values of cells in the G1-phase. Thus statistical tests for a shift in the nuclear DNA content of the cells in question in the G1-phase from that of control cells were made possible.

Cell Cycle↗