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PubMed1991

[FTIR-microspectrophotometry for high resolution and highly sensitive detection of the carboxyhemoglobin complex].

Our recently developed method for rapid quantitative evaluation of carboxyhemoglobin in liquid as well as in dried blood samples has been considerably improved by the incorporation of an infrared microscope. Besides the characteristic bands at V = 1953 (cm-1) in liquid and V = 1968 (cm-1) in dry blood we found, upon addition of D2O, a dislocation of the bands at V = 1949 (cm-1). This reversible dislocation still permits a reliable identification of the COHb complex at the extremes of the capacity range of the measuring unit. Thus identification of the COHb complex was successful even in isolated erythrocytes. By means of this improved technique reliable quantitative evaluation may be carried out in a concentration range of between 2% and 0.05% whereas semiquantitative evaluation was possible at concentration down to 0.01%, allowing for instance the evaluation of the effects of a single inhalation of cigarette smoke. The positive identification of endogenous COHb fractions as well as of exogenous agents (smoking, environmental factors) which this method permits is prerequisite for a dependable interpretation of forensic-toxicologically relevant COHb findings.

Carboxyhemoglobin↗
PubMed1991

The clinical usefulness of determining ploidy patterns in human tumors as measured by slide-based Feulgen microspectrophotometry.

Some aspects of the clinical value of the Feulgen microspectrophotometric assessment of DNA ploidy patterns in human tumors are reviewed. This method has been shown to be of predictive value for a number of tumor sites and may be independent of other prognostic indicators, such as the histopathologic grade. The association between ploidy and prognosis probably reflects the degree of chromosomal changes in the tumor cells; while it is probable that all malignant tumors are aneuploid, there is a tendency for the changes to be more extensive in more aggressive tumors. Thus, tumors with DNA modes that depart significantly from the diploid and tetraploid levels may have a worse prognosis than do tumors whose modes are at or close to these levels. This has clearly been shown for tumors of the breast, ovary, endometrium and several other sites. For some sites, including the cervix uteri and the large bowel, such a relationship is less clear, probably because tumors at these sites have frequently undergone extensive chromosomal changes that do not result in a significant deviation of the DNA mode from the euploid levels. The use of slide-based DNA analysis systems, in which the morphology of the cells being measured can be assessed, has advantages over flow cytometry that may be crucial in some situations.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosomes↗
PubMed1976

Perspectives on the use of microspectrophotometry in the diagnosis of pretumorous processes and malignant tumors.

Comparative microspectrophotometric study of the nuclear Feulgen DNA content in pretumorous processes and cancers (larynx, endometrium, large intestine, and stomach) has made it possible to formulate an exponential law of DNA accumulation in cell populations in proliferating and malignant tissue. The dynamics of changes in the "index of DNA accumulation" can be used as an objective quantitative diagnostic test. A scale of malignant transformation was proposed with which the degree of probability of malignancy in pretumorous processes could be estimated. The data obtained allowed the author to propose a hypothesis of DNA superproduction as the basic cause of progressive neoplastic growth.

Chromosomes↗
PubMed1976

Selection of operating wavelength-pair for two-wavelength microspectrophotometry.

The two-wave length method is the microspectrophotometric method of choice for thick specimens. Its accuracy is dependent on the accuracy of selection of the operating wavelenght-pair. The "slope zero test", offered as a check upon the accuracy of this selection (Garcia and Iorio, '66) is shown to be invalid by analysis of experimental data, by computer model and by mathematical derivation. A means of checking the accuracy of selection of the operating wavelength-pair via scanning measurements is suggested. A new formulation of the two-wavelength method, permitting use of different operating wavelength-pairs, is also given.

Absorption↗
PubMed1986

Kinetic analysis in single, intact cells by microspectrophotometry: evidence for two populations of erythrocytes in an individual heterozygous for glucose-6-phosphate dehydrogenase deficiency.

A microspectrophotometric technique was used to measure the kinetics of methemoglobin reduction in intact, unaltered human erythrocytes. Reduction was catalyzed by endogenous NADPH-methemoglobin reductase in the presence of Nile Blue. The technique was applied to the study of erythrocytes from a female donor with decreased glucose-6-phosphate dehydrogenase (G-6-PD) activity. The individual was shown to be heterozygous for deficiency of G-6-PD. The kinetic study revealed two distinct populations of erythrocytes that were nearly equal in number. One cell population showed reduction rates between 0 and 25% of normal, whereas the second cell population displayed rates within the range seen for normal cells. Single-cell indices of cell size, cell hemoglobin content, and ratio of cell hemoglobin to cell size did not correlate with single-cell reduction rates and were not significantly different between the two populations. These results provide quantitative support for the X-inactivation hypothesis in G-6-PD deficiency.

Adult↗
PubMed1986

DNA microspectrophotometry of bone sarcomas in tissue sections as compared to imprint and flow DNA analysis.

The aim of the present study was to establish an upper limit of diploidy for microspectrophotometric (MSP) DNA measurements in sections of mesenchymal tissue analyzing DNA data of a large number of normal cell populations. The reliability of this upper limit of diploidy for discriminating between diploid and hyperploid bone sarcomas was tested by analyzing the same tumors by MSP in imprint preparations and flow cytometry (FCM). The median DNA value of control cells in tissue sections was given arbitrary value of DNA index (DI) 1.0, denoting the diploid DNA content. The proportion of cells with DNA values exceeding DI 1.25 (greater than DI 1.25) was determined for each normal cell population. The maximum percentage of cells with DNA values exceeding DI 1.25, encountered by analysis of 91 normal cell populations in tissue sections, was 31%. This percentage was set as an upper limit of diploidy. Hence, tumors with a higher percentage of cells greater than DI 1.25 were classified as hyperploid. When we applied this criterion, 31 of 36 sarcomas analyzed by MSP in tissue sections were hyperploid, which was in complete agreement with FCM and MSP in imprints of the same tumors. Apart from discriminating between diploid and hyperploid tumors, an attempt was made to determine peak DNA values of sarcomas analyzed in tissue sections. Peak DNA values, as defined by a minimum of 30% of the cells within a class width of DI 0.25, could be determined for 23 of 36 tumors. These peak DNA values correlated well with corresponding peaks obtained by FCM.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Neoplasms↗
PubMed1984

Blue-sensitive cones in the primate retina: microspectrophotometry of the visual pigment.

Direct absorbance and bleaching absorbance-difference spectra were obtained using a photon-counting microspectrophotometer from the outer segments of ten blue-sensitive cones of macaque monkeys. The peak wavelength (lambda max) of the direct measurements was 426 +/- 3.4 nm, whereas the lambda max of the bleaching difference was 434 +/- 6.6 nm. We consider these values to be upper and lower bounds since both measurements may be shifted in opposite directions by wavelength-dependent effects. Therefore, the true peak sensitivity must be close to 430 nm.

Animals↗
PubMed1981

Quantitation of catalase activity by microspectrophotometry after diaminobenzidine staining.

The absorbance of the reaction product of catalase staining with diaminobenzidine is linearly proportional to enzyme activity. This is shown in semithin Epon sections of model systems containing serum albumin and catalase from bovine or guinea pig liver. Absorbance measurements were also performed on semithin sections of guinea pig liver, and from these, the activity of cytoplasmic (extraperoxisomal) catalase has been derived.

3,3'-Diaminobenzidine↗
PubMed1976

Microspectrophotometry of mitochondrial cytochrome P-450 in single adrenal cells.

The cytochrome P-450 is known to be a key enzymic component in steroid hydroxylation. Biochemically it is localized in adrenal cells, both in the mitochondrial and in the microsomal fractions. Owing to its characteristic light absorptivity, attempts were made at its localization and measurement in a microspectrophotometric system. Specific difference spectra were obtained in the cytoplasm of some cells from an adrenal cell suspension before and after saturation with carbon monoxide. Scanning at 450 nm showed slender absorption maxima after CO saturation randomyl distributed in the cytoplasm. These may be attributed to mitochondria or larger cytoplasmic functional units including mitochondria.

Adrenal Glands↗
PubMed1982

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↗
PubMed2005

Visual pigment spectra of the comma butterfly, Polygonia c-album, derived from in vivo epi-illumination microspectrophotometry.

The visual pigments in the compound eye of the comma butterfly, Polygonia c-album, were investigated in a specially designed epi-illumination microspectrophotometer. Absorption changes due to photochemical conversions of the visual pigments, or due to light-independent visual pigment decay and regeneration, were studied by measuring the eye shine, i.e., the light reflected from the tapetum located in each ommatidium proximal to the visual pigment-bearing rhabdom. The obtained absorbance difference spectra demonstrated the dominant presence of a green visual pigment. The rhodopsin and its metarhodopsin have absorption peak wavelengths at 532 nm and 492 nm, respectively. The metarhodopsin is removed from the rhabdom with a time constant of 15 min and the rhodopsin is regenerated with a time constant of 59 min (room temperature). A UV rhodopsin with metarhodopsin absorbing maximally at 467 nm was revealed, and evidence for a blue rhodopsin was obtained indirectly.

Animals↗
PubMed1999

A rapid wavelength changer based on liquid crystal shutters for use in ratiometric microspectrophotometry.

Many of the fluorescent indicator molecules most useful for measuring intracellular concentrations of ions of biological importance, such as Ca2+ or H+, require illumination first at one wavelength, at which the fluorescence depends strongly on the concentration of the ion, and then at another wavelength (e.g. the isosbestic point), so that a ratio can be obtained. Existing wavelength changers are mechanical and involve moving filters, mirrors or gratings. These systems have the disadvantage that they introduce mechanical shocks that can interfere with simultaneous electrophysiological recording. In addition, they require special electrical driving systems and are relatively expensive, especially if they are capable of switching rapidly. We describe a new wavelength changer based on liquid crystal shutters which has the following advantages: (1) it has no mechanical moving parts; (2) it can switch rapidly (@1 ms) and in any desired pattern (off - on1 - off - on2 - off, etc.); (3) it is driven by a low-power 15-V pulse; and (4) it is substantially cheaper than existing wavelength changers. Its limitations are that it does not pass wavelengths shorter than about 400 nm and transmission in the range 430-700 nm is only 20-40%.

Calcium↗