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PubMed1982

Kinetics of reactions within single erythrocytes: studies by microspectrophotometry.

Single cell spectroscopy has been applied to red blood cells along three different lines of research: a) distribution of single hemoglobin components among different erythrocytes, b) chemical reaction with ligands and diffusion in the red blood cell c) dynamics of hemoglobin S aggregation in single red blood cell. The results reported here show, in general, the great possibilities of using single-cell spectroscopy in investigating complex integrated systems.

Anemia, Sickle Cell↗
PubMed1976

Quantitative cytochemistry of glycogen in blood cells. Methods and clinical application.

Quantitative glycogen determinations can be made in single blood and bone marrow cells, using microspectrophotometry or microfluorometry after staining with variants of the periodic acid--Schiff (PAS) reaction. These PAS variant reactions generally do not indicate the presence of non-glycogen PAS-positive substances, known to be prevalent in various hematopoietic cells, possibly due to masking of reactive groups. The specificity of the reaction in blood cells was ascertained by alpha-amylase digestion, which removed more than 95% of the PAS-positive material. Calibration of the PAS reaction was undertaken with a microdroplet model of pure leukocyte glycogen. The glycogen amounts in the droplets were determined by microinterferometry, the droplets were stained with a variant PAS reaction, and the total extinction of the reaction product in the stained droplets was determined by microspectrophotometry. The extinction coefficient (k) was obtained from the equation k equals Etot divided by M where (Etot) is the total extinction as determined by microspectrophotometry and (M) the dry glycogen amount as determined by microinterferometry. The microinterferometric dry mass determinations were calibrated by X-ray absorption in order to obtain the absolute amounts of glycogen. For practical purposes a reference system was made of normal neutrophil leukocytes. The glycogen content in the reference neutrophils was first determined with the micromodel. These neutrophils, now with a known glycogen amount, were stained with the PAS reagents and measured microspectrophotometrically in parallel with cells containing an unknown glycogen amount. Alternatively, the staining was made with a fluorescent PAS reaction, and the glycogen content determined by microfluorometry. Both methods appeared suitable for determining the glycogen content of blood cells from patients with various diseases, though the microfluorometric method was preferable for measurements of small amounts of inhomogeneously distributed glycogen. The mean glycogen content of normal neutrophil leukocytes was found to be 13.6 times 10(-12) g. The content was increased in infectious diseases such as pneumonia and tonisillitis, as well as in polycythemia vera and myelofibrosis, while low amounts were found in untreated chronic myelocytic leukemia. In chronic myelocytic leukemia in remission, the glycogen content of mature neutrophils had completely normalized. Erythroblasts normally do not contain detectable amounts of glycogen. However, in certain diseases such as beta-thalassemia and Di Guglielomo's syndrome, appreciable amounts of glycogen accumulate in the erythropoietic precursor cells. In beta-thalassemia this was associated with an arrest in the proliferation of early polychromatic erythroblasts, which accumulate glycogen in the G1 phase of the cell cycle. In all these diseases quantitative glycogen determinations in the blood cells have diagnostic importance.

Autoradiography↗
PubMed1989

DNA evaluation in growth hormone producing pituitary adenomas: flow cytometry versus single cell analysis.

DNA patterns were analysed in 26 GH-producing pituitary adenomas by flow cytometry as well as by microspectrophotometry. Twelve tumours (46%) were diploid according to both methods, whereas 5 tumours (19%) showed aneuploid DNA patterns. Nine tumours were classified differently by the two methods: flow cytometry resulted in diploidy in 2 and aneuploidy in 7 patients, whereas microspectrophotometry showed diploidy in 5 tumours, tetraploidy in 3 and aneuploidy in 1. Methodological limitations may explain the discrepancy in the results obtained by the two methods. However, both the flow cytometry and the microspectrophotometry method show the presence of aneuploid DNA patterns in GH-producing pituitary adenomas despite their benign growth characteristics and the clinically benign course of the disease. This comparative study with two methods measuring DNA content, shows that depending on the criteria used for diploidy-aneuploidy, the frequency of aneuploidy will vary. In this material of 26 GH-producing adenomas, 46% were aneuploid according to flow cytometry and 23% according to microspectrophotometric. However, no correlation to tumour size or GH levels was found with either method when patients with aneuploid and diploid tumours were compared. Therefore, no clinical significance can so far be drawn from these results.

Acromegaly↗
PubMed1989

Ploidy and morphology in osteosarcoma.

In a cytometric DNA study of high-grade osteosarcoma, the relationship between DNA content and morphology was analyzed. The investigation, based on microspectrophotometry of tissue sections and flow cytometry (FCM), included both primary lesions and recurrences. FCM analysis, applied to a consecutive series of 47 primary osteosarcomas, disclosed that 2 were diploid and 45 were nondiploid, 8 of which were tetraploid. Multiple aneuploid peaks were detected in 13 tumors. Among the nondiploid tumors, there was no clear relationship between the peak DNA value(s) and the histologic subtype (osteoblastic, chondroblastic, fibroblastic) or grade (III-IV). The proliferative activity, as reflected by the percentage of S-phase cells, could be determined in 38 of the 47 tumors analyzed by FCM. The percentage was higher for aneuploid than for tetraploid lesions; however, the distribution of S-phase cells was not related to the histologic subtype or the grade of the tumors. To assess the reliability of a single sample for FCM, the DNA content of biopsy and surgical specimens was compared in 20 tumors; there was complete agreement in all cases with respect to the classification of the lesion as diploid, tetraploid or aneuploid. Analysis by FCM or microspectrophotometry of 12 local recurrences and 16 metastases and the corresponding 19 primary tumors showed that an aneuploid characteristic of the primary lesion was retained during progression of the disease. In 12 tumors analyzed by microspectrophotometry in tissue sections, comparison of chondroblastic and osteoblastic/fibroblastic areas within the same lesion consistently disclosed hyperploidy in both areas.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Neoplasm↗
PubMed1998

[Intratumoral heterogeneity of DNA content in lung adenocarcinoma].

Quantitative microspectrophotometry was performed in 10 resected lung adenocarcinomas for the purpose of studying intratumoral heterogeneity of DNA content. Histologically, there were 3 stage I, 2 stage IIIA, 2 stage IIIB, and 3 stage IV cases, consisting of 3 well, 4 moderately and 3 poorly differentiated cases. The tumors were cut at the greatest dimension and fine needle aspirations were performed from 5 separate areas (central, cranial, caudal, inward, and outward). One of the imprint smears was submitted for cytology and the other for microspectrophotometry using Feulgen staining. Although G0G1 and G2M phase cell populations varied in each specimen, all 5 specimens showed the same DNA histogram in 9 cases. These were 4 aneuploid, 4 polyploid (euploid but not diploid) and only one diploid tumor. Two stem lines were observed in the remaining single case. One of 5 specimens showed aneuploid, the other 4 specimens showed polyploid. Namely, intratumoral heterogeneity was observed in one case. Ther was no relationship between intratumoral heterogeneity tumor size or differentiation. Most lung adenocarcinomas showed DNA stability. Fine needle aspiration specimens obtained from one area of lung adenocarcinoma represent the DNA contents of the entire tumor.

Adenocarcinoma↗
PubMed1992

The eyespot of Euglena gracilis: a microspectrophotometric study.

The eyespots in cells of streptomycin-bleached strains and of dark-grown cultures of Euglena gracilis, were examined by means of fluorescence microscopy and microspectrophotometry. When viewed with light in the region of 380-500 nm, the stigma appeared as a dark spot. Adjacent to this was a second spot, not seen with white light, but which was seen to fluoresce when excited with radiation at 370 +/- 20 nm. This fluorescence proved to be polarized in contrast to other fluorescing bodies in the cell. The absorption curves, obtained by microspectrophotometry of individual eyespots, were found to consist of two spectral maxima, an A-band in the blue and a B-band in the green. Unlike the A-band, the B-band provided evidence of originating from an anisotropic structure. Relating these data to literature findings, we conclude that the B-band is the absorbance of a pigment in the quasi-crystalline paraflagellar body and the A-band perhaps a pigment in the orange-red stigma. The spectrum of the B-band does not appear to be that of a flavoprotein or of a free carotenoid but its resemblance to the spectrum of rhodopsin is significant in relation to published data for the Chlamydomonas eyespot that suggests the presence of a rhodopsin-like pigment as the photosensitive system responsible for phototaxis in this alga.

Animals↗
PubMed2005

Topochemical studies on modified lignin distribution in the xylem of Poplar (Populus spp.) after wounding.

BACKGROUND AND AIMS: Information on the influence of wounding on lignin synthesis and distribution in differentiating xylem tissue is still scarce. The present paper provides information on cell modifications with regard to wall ultrastructure and lignin distribution on cellular and subcellular levels in poplar after wounding. METHODS: Xylem of Populus spp. close to a wound was collected and processed for light microscopy, transmission electron microscopy and cellular UV microspectrophotometry. Cell wall modification with respect to lignin distribution was examined at different stages of wound tissue development. Scanning UV microspectrophotometry and point measurements were used to determine the lignin distribution. KEY RESULTS: Xylem fibres within a transition zone between differentiated xylem laid down prior to wounding and the tissues formed after wounding developed distinctively thickened secondary cell walls. Those modified walls and cell corners showed, on average, a higher lignin content and an inhomogeneous lignin distribution within the individual wall layers. CONCLUSIONS: The work presented shows that wounding of the xylem may induce a modified wall architecture and lignin distribution in tissues differentiating at the time of wounding. An increasing lignin content and distinctively thickened walls can contribute to improved resistance as part of the compartmentalization process.

Cell Differentiation↗