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PubMed2006

High-performance liquid chromatography-ultraviolet-visible spectroscopy-electrospray ionization mass spectrometry method for acrylic and polyester forensic fiber dye analysis.

A critical point of comparison between a fiber collected from a crime scene and a fiber from a known source is the color. Fiber dye analysis using thin-layer chromatography or ultraviolet (UV)-visible (Vis) microspectrophotometry provides useful, although limited, data for comparison. High-performance liquid chromatography-electrospray ionization mass spectrometry (LC/MS) overcomes these limitations by integrating chromatography, ultraviolet-visible spectroscopy, and mass spectrometry into a single instrument. In order to evaluate the applicability of the LC/MS to forensic fiber dye analysis, a multi-stage chromatographic method using acidified water and acidified acetonitrile was developed that separated and identified a mixture of 15 basic and 13 disperse dye standards. The LC/MS also detected and analyzed dyes extracted from individual 0.5 cm acrylic and polyester fibers, demonstrating its applicability to this type of analysis. With regard to the analysis of disperse dyes in polyester fibers, the replacement of pyridine with acetonitrile in the extraction system allowed direct injection of the extracts into the LC/MS. The advantage of the LC/MS over other instrumental methods of textile dye analysis is demonstrated by the analysis and differentiation of three black acrylic fibers: two fibers had similar UV-Vis spectra but were differentiated with chromatography and two had similar UV-Vis spectra and chromatograms but were differentiated using the mass spectrometer.

Journal Article↗
PubMed1982

Cell cycle distribution of erythroblasts in P. falciparum malaria.

The cell cycle distribution of the erythroblasts of 6 Gambian children with P. falciparum malaria was determined using the technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography. The data showed evidence of a perturbation of erythroblast kinetics in all 6 children. The abnormalities were more marked in the children with chronic malaria than in those with acute malaria and predominantly affected the early polychromatic erythroblasts. The disturbances observed in chronic malaria included an increased proportion of cells in the G2 phase and an arrest during the progress of some cells through the S phase. The latter defect was not seen in the children with acute malaria.

Acute Disease↗
PubMed1986

Glycogen content and PAS staining pattern of human megakaryocytes.

The glycogen content of human megakaryocytes was studied using a quantitative method. Smears of bone marrow from 13 individuals were stained with the modified PAS reaction with and without prior treatment with alpha-amylase. The intensity of the reaction was determined by microspectrophotometry in 50 megakaryocytes from each individual. It was found that megakaryocytes are rich in glycogen which is not only confined to the intensely PAS-positive granules and inclusion bodies, but also makes up a good part of the diffuse cytoplasmic staining. In the diffusely stained megakaryocytes, glycogen makes up 32% of the intensity of the PAS reaction, while it reaches 47% in those with granules and up to 60% in those with inclusion bodies. The total extinction of the PAS-stained megakaryocytes is not only dependent on the morphological appearances of the cells, but is also positively correlated with their size.

Bone Marrow Examination↗
PubMed1987

Quantitation of hemoglobin in single erythrocytes with and without fetal hemoglobin.

The hemoglobin content was determined by microspectrophotometry in single erythrocytes with and without fetal hemoglobin (Hb F) from 16 normal subjects, 30 patients with anemia of different etiology and severity and 20 individuals with thalassemic disorders. Hb F-containing cells were identified by an indirect immunofluorescent method. The relative single cell value: total extinction (TE) at 415 nm, cell size (A) and the ratio TE/A, were used to indicate single cell values of MCH, MCV and MCHC respectively. The TE (MCH) and/or TE/A (MCHC) did not differ significantly between Hb F-containing (F-cells) and non-F cells in normal subjects and in cases with various forms of acquired anemia. On the contrary, the TE and/or TE/A of F-cells was found significantly higher in F compared to non-F cells in 11 of 20 (55%) of the cases with thalassemia. The results suggest that, in some cases of thalassemia, hemoglobin F is an important substitute for hemoglobin A and may improve the level of hemoglobinization.

Erythrocytes↗
PubMed1985

DNA content of hepatocytes in various stages of liver cirrhosis.

In order to search for some parameters that would make it possible to predict a potentially high risk of evolution from liver cirrhosis to hepatocellular carcinoma (HCC), the DNA content of hepatocytes in patients with liver cirrhosis with or without development to HCC was investigated by means of microspectrophotometry in Feulgen-stained specimens. In patients without development of HCC in more than 5 years of follow-up, 90% or more of tested hepatocytes were diploid, whereas in patients with liver cirrhosis who developed HCC within 3 years the frequency histogram of nuclear DNA content was widely spread from diploid to hyperpolyploid with a small peak of triploid. Furthermore, in the latter group of patients, many of the binucleate cells had different DNA contents in each paired nucleus. In non-cancerous portions of HCC, the ploidy histogram of nuclear DNA content was widely spread from diploid to polyploid with different DNA contents of each paired nucleus in binucleate cells, without the small peak of triploid. These results suggest that the deranged cell kinetics of hepatocytes in cirrhosis may be considered to be a state of potentially high risk for the evolution of HCC, and that a patient with liver cirrhosis with such an abnormal hepatocyte DNA content should be followed up carefully for the early diagnosis of HCC.

Adult↗
PubMed1975

Cytokinetic variations during ageing and regenerative growth in the jb-1 ascites tumour studied by impulse cytophotometry.

The variation in the DNA distributions in the murine JB-1 ascites tumour was investigated by means of impulse cytophotometry (ICP) on days 2, 4, 7, 10 and 14 after i.p. inoculation of 2.5 times 10-6 tumour cells and on days 1, 2, 3 and 4 after aspiration of the main part of 10-day plateau tumours. The study showed that a considerable fraction of cells with a DNA content of about 2 and 4 n (p smaller than 0.001) accumulated from day 2 to 10. During the same period the fraction of cells in S decreased significantly (p smaller than 0.001). One and two days after aspiration, a rapid rearrangement in the distribution pattern occurred, leading to a predominance of cells with S DNA content, while the fraction of cells around 2 n and 4 n decreased. Three and four days after aspiration the cells again accumulated around the 2 and 4 n values while the fraction of cells with S DNA content decreased. The ICP results were in agreement with our data obtained by autoradiography and Feulgen microspectrophotometry, and ICP is recommended as a rapid and statistically precise method in cytokinetic analysis.

Animals↗
PubMed1992

DNA content in primary tumours and lymph node metastases in colorectal adenocarcinoma.

In 18 consecutive patients operated on for colorectal carcinoma of Dukes' stage C, the DNA patterns were determined in multiple samples of the primary tumours and in all detected lymph node metastases. Single-cell microspectrophotometry on Feulgen-stained smears of fine-needle aspirates was used. When the most aggressive DNA pattern was considered representative, 12 primary tumours (67%) were designated as aneuploid. The frequency of aneuploidy among the metastases was almost the same (63%). In 15 cases (83%) the DNA patterns displayed by the metastatic lymph nodes were also found in the corresponding primary tumour, while in the remaining three cases (17%) the DNA pattern in the lymph node metastases was not seen in any of the multiple samples from the primary tumour. The observed tumour DNA heterogeneity may reflect either the multicellular origin of the tumour cells or the continuous evolution and progression of a neoplasm of unicellular origin, and may partly explain the dissimilarities between the DNA patterns of the primary tumour and the lymph node metastases. Biopsy samples from a number of metastatic lymph nodes are therefore required to ensure representativeness and to permit an adequate analysis of the prognostic role of the DNA ploidy status in lymph node metastases from colorectal cancer.

Adenocarcinoma↗
PubMed1994

Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.

ERG mass photoreceptor responses were recorded across the isolated, aspartate-perfused retina of the frog, Rana temporaria, in order to determine spectral sensitivities of cones. Cone responses were distinguished from rod responses by their faster kinetics, and responses from different cone types were isolated by selective background adaptation. Our main finding is that of a novel short-wavelength sensitive cone population peaking at about 431 nm. Further, we find that the sensitivity spectrum of the dominant long-wavelength sensitive cone population fully accounts for the most common type of photopic ganglion cell spectrum. Both can be described by a nomogram with lambda max = 562 nm. This resolves a long-standing apparent conflict between cone absorbance spectra and ganglion cell sensitivities. Including the 502 nm cones previously described by microspectrophotometry, the frog possesses a collection of cones that could support trichromatic photopic vision.

Animals↗
PubMed1992

Photobleaching difference absorption spectra of human cone pigments: quantitative analysis and comparison to other methods.

Four human cone pigment apoproteins were expressed by transfection of human tissue culture cells with the corresponding complementary DNA clones. Following reconstitution of the cone pigments by incubation with 11-cis retinal, photobleaching difference absorption spectra were obtained for the blue pigment, the green pigment, and two polymorphic variants of the red pigment. These spectra were analyzed to determine the wavelengths of maximal absorbance and the bandwidths. The recombinant cone pigment spectra were compared to human cone spectral sensitivities and cone pigment absorption spectra determined by microspectrophotometry, single-cell electrophysiology, reflection densitometry, electroretinography, and psychophysical color and brightness matching.

Cloning, Molecular↗
PubMed1973

The optical density of erythrolabe determined by retinal densitometry using the self-screening method.

1. Retinal densitometry has been used to determine the optical density of erythrolabe in foveal cones.2. A self-screening method was used, as in Rushton's (1963b) measurements on chlorolabe.3. Psychophysical experiments have demonstrated that self-screening in cones depends on the direction of the light in the same manner as the Stiles-Crawford efficiency. Stiles-Crawford measurements were therefore made on each subject and a method was devised to allow for the corresponding reduction in self-screening in densitometry.4. Complications arising from coloured bleaching products, the presence of a second visual pigment and the fluorescence of visual pigments are considered.5. The mean value for optical density at 560 nm was found to be 0.40 +/- 0.07 S.E. of mean for the five subjects (two deuteranopes, two deuteranomalous subjects and one normal subject).6. This result is in good agreement with Rushton's (1963b) value of 0.35 for chlorolabe using retinal densitometry.7. The densitometry results are also in reasonable accord with recent psychophysical and microspectrophotometry measurements. This provides evidence that densitometry measures all the cone pigment in an unbiased manner and not just a superficial sample.

Color Vision Defects↗
PubMed1973

The optical density of erythrolabe determined by a new method.

1. A new method is described for the determination of optical density by retinal densitometry based on an analysis of stray light.2. The basic assumption used is that the stray light present in retinal densitometry is independent of wave-length. The justification for this assumption is considered.3. The stray light and hence the optical density was determined by a method of successive approximations using a Linc 8 computer.4. The optical density of erythrolabe for the direction of maximum Stiles-Crawford efficiency was determined for five subjects, two deuteranopes, two deuteranomalous and one red-rich normal subject. The mean values for three of the subjects were close to the grand mean value of 0.42, but the other two subjects had considerably higher and lower values respectively.5. The results are in satisfactory agreement with optical density values determined by the self-screening method using retinal densitometry, and also with determinations using microspectrophotometry and psychophysics.6. The relevance of dielectric wave-guide modes in the outer segments is considered.

Color Vision Defects↗
PubMed1979

Pigment transformation and electrical responses in retinula cells of drone, Apis mellifera male.

1. Receptor potentials in honeybee drone retinula cells were recorded with intracellular micro-electrodes in the dorsal part of the superfused retina. The light stimuli were sufficiently weak that the response amplitude was proportional to the intensity. 2. Responses to stimuli of different wave-lengths, although of different amplitude, all had the same time course. 3. The maximal sensitivity in all the cells recorded from was to a wave-length between 450 and 460 nm. 4. Microspectrophotometry showed the presence of a pigment with two stable states, interconvertible by light, absorbing maximally at 445 nm (rhodopsin) and 505 nm (metarhodopsin). 5. There was a good match between the absorption spectrum of rhodopsin and the spectral sensitivity of retinula cells. 6. Transformation of a large fraction of rhodopsin to metarhodopsin by light reduced the sensitivity of the retinula cell but did not alter the shape of the relative spectral sensitivity curve or the time course of the responses. 7. It is concluded that for weak lights the receptor potential is determined only by the number of rhodopsin molecules that absorb photons: neither the presence of metarhodopsin nor its phototransformation to rhodopsin produces a detectable effect.

Animals↗
PubMed1980

Electrical responses and photopigments of twin cones in the retina of the walleye.

1. The properties of twin and single cones in the retina of the walleye (Stizostedion vitreum vitreum) were studied by intracellular recording, dye injection and microspectrophotometry. 2. Twin cones generate hyperpolarizing responses to central illumination, can receive depolarizing influences (feed-back) from the receptive field surround, and show no detectable dye coupling when injected with Procion yellow. In seventeen of eighteen dye-injected cones, fluorescence was intense in the inner segment and undetectable or weak in the cone pedicle. 3. Both members of the twin cone contain the same photopigment in their outer segments. It absorbs maximally at about 605 nm. 4. A 533 nm green-sensitive photopigment was found in single cones. No blue-sensitive cones have been found. 5. With the exception of a modest discrepancy in the violet, the absorptance spectrum of the 605 nm photopigment of twin cones agrees closely with the action spectrum measured by intracellular recording. 6. The spectral properties established by the twin cone's photopigment are not detectably altered by the hyperpolarizing influences arising from nearby cones or by the depolarizing influences arising from the receptive field surround. 7. The twin cones of the walleye retina are thus "identical twins', both photochemically and physiologically, and seem designed to function as long-wave, spectrally univariant receptor units for colour vision. 8. The available evidence suggests that identical twin cones differ functionally from double cones and non-identical twin cones. 9. Although they outnumber single cones by about three to one in adults, identifiable twin cones were rarely observed in the cone population of retinas examined 3-5 days after birth. If walleye twin cones develop by fusion of single cones this process apparently occurs only for cones containing the 605 nm photopigment.

Animals↗
PubMed1990

The frequency of isomerization-like 'dark' events in rhodopsin and porphyropsin rods of the bull-frog retina.

1. The dark current and responses to dim flashes were recorded with the suction pipette technique from single rods in pieces of bull-frog retina taken from either the dorsal porphyropsin or the ventral rhodopsin field. 2. The composition of visual pigment in the rods was determined by microspectrophotometry. Rods from the dorsal pieces contained 70-88% porphyropsin523 mixed with rhodopsin502. The ventral rods contained almost pure rhodopsin, any possible admixture of porphyropsin being below the level of detectability (less than 5%). 3. In most cells, the responses to dim flashes were well fitted by a four-stage linear filter model, with no systematic differences in the response kinetics of porphyropsin and rhodopsin rods. The amplitude of saturated responses varied between 8 and 55 pA and that of responses to single isomerizations between 0.4 and 3.5 pA. 4. In porphyropsin rods, discrete events similar to the response to one photoisomerization were clearly seen in complete darkness. The dark current amplitude histogram was fitted by a convolution of the probability densities for the Gaussian continuous noise component and the averaged dim-flash response waveform. This allows estimation of the frequency and amplitude of discrete events and the standard deviation of the continuous component. The mean frequency of discrete dark events thus obtained from six porphyropsin cells was 0.057 rod-1 s-1 at 18 degrees C. 5. In rhodopsin rods, the dark current amplitude histogram appeared completely symmetrical, indicating that the frequency of discrete events must be lower than 0.005 rod-1 s-1 (except in one rod where it was 0.006 events rod-1 s-1). Per molecule of rhodopsin, the events are then at least 5 times rarer than reported for toad rhodopsin rods at the same temperature. 6. The low rate of isomerization-like 'dark' events in bull-frog rhodopsin rods shows, firstly, that results cannot be generalized across species even for rhodopsins which appear spectrally identical. Secondly, it suggests that these events need not (in an evolutionary sense) constitute an irreducible noise factor which must set the ultimate limit to the sensitivity of dark-adapted vision. 7. The difference between porphyropsin and rhodopsin rods shows that, given (presumably) the same opsin, the pigment utilizing retinal2 and absorbing maximally at longer wavelengths produces more noise. The signal/noise ratio attained in the photoreceptor may be an important factor in the natural selection of visual pigments.

Animals↗
PubMed2002

pH and rate of "dark" events in toad retinal rods: test of a hypothesis on the molecular origin of photoreceptor noise.

Thermal activation of the visual pigment constitutes a fundamental constraint on visual sensitivity. Its electrical correlate in the membrane current of dark-adapted rods are randomly occurring discrete "dark events" indistinguishable from responses to single photons. It has been proposed that thermal activation occurs in a small subpopulation of rhodopsin molecules where the Schiff base linking the chromophore to the protein part is unprotonated. On this hypothesis, rates of thermal activation should increase strongly with rising pH. The hypothesis has been tested by measuring the effect of pH changes on the frequency of discrete dark events in red rods of the common toad Bufo bufo. Dark noise was recorded from isolated rods using the suction pipette technique. Changes in cytoplasmic pH upon manipulations of extracellular pH were quantified by measuring, using fast single-cell microspectrophotometry, the pH-dependent metarhodopsin I-metarhodopsin II equilibrium and subsequent metarhodopsin III formation. These measurements show that, in the conditions of the electrophysiological experiments, changing perfusion pH from 6.5 to 9.3 resulted in a cytoplasmic pH shift from 7.6 to 8.5 that was readily sensed by the rhodopsin. This shift, which implies an 8-fold decrease in cytoplasmic [H(+)], did not increase the rate of dark events. The results contradict the hypothesis that thermal pigment activation depends on prior deprotonation of the Schiff base.

Animals↗
PubMed1959

Redox absorption spectra from single pigment cells of squid.

Single pigment cells from the squid Loligo forbesi have been studied by microspectrophotometry. The absorption spectra obtained show characteristic changes on reduction and oxidation which are compatible with those found in ommochromes. The presence of melanoid substances, however, cannot be excluded.

Animals↗
PubMed1964

DIPLOID AND ENDOREDUPLICATED CELLS: MEASUREMENTS OF DNA.

Two-wavelength microspectrophotometry has shown that endoreduplicated cells in an x-irradiated culture of human leukocytes contained about twice as much DNA as diploid cells from the same culture. This supports the assumption that endoreduplicated cells represent a form of polyploidy.

Chromosomes↗