From cytofluorometry to fluorescence image analysis.

Urata, Yoji; Itoi, Hirosumi; Murata, Shin‐ichi; Konishi, Eiichi; Ueda, Kazushige; Azumi, Yuji; Ashihara, Tsukasa

doi:10.1267/ahc.24.367

Cited by 5 publications

(4 citation statements)

References 9 publications

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“…We have reported that cell cycle and the ploidy pattern of the tumor affected nuclear morphology (Murata, 1991;Murata et al, 1993bMurata et al, , 2001a). Nonthyroid tumors with nondiploid ploidy patterns usually show prominent nuclear atypia Urata et al, 1991), whereas thyroid cancers usually show diploid ploidy pattern and slow growth with a lack of marked cellular atypia (Murata, 1991;Murata et al, 1989Murata et al, , 1990. However, Hashimoto's disease, adenomatous hyperplasia, and tumors often include large bizarre cells scattered among cells with low-grade nuclear atypia.…”

Section: Discussionmentioning

confidence: 99%

Morphological abstraction of thyroid tumor cell nuclei using morphometry with factor analysis

Murata

Mochizuki

Nakazawa

et al. 2003

Microscopy Res & Technique

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Various morphonuclear studies by digital image analysis have successfully been applied to quantify the nuclear morphology, including chromatin distribution pattern, in cytology of various organs; however, the majority of past reports have not shown correlation between the quantitative data by digital image analysis and cytological findings in practical diagnosis. In this report, we present the usefulness of morphological abstraction to combine the objective data and subjective observation in cytological diagnosis. Randomly selected, 100 cells in each Papanicolaou-stained ABC smear samples of 39 benign and malignant thyroid tumor cases were studied. Gray-level image data provided seven parameters for nuclear size, four parameters for nuclear shape, and 16 parameters showing the nuclear chromatin patterns from high-dimensional texture analysis of using co-occurrence and run-length matrices. To statistically abstract nuclear morphology, factor analysis was used. Factor analysis classified morphological nuclear characters as abstraction parameter into five abstract parameters composed of nuclear size, shape, heterogeneity, and contrast and homogeneity of chromatin pattern. The nuclei of papillary carcinoma showed larger size, more irregular shape, and higher contrast of chromatin pattern than those of the benign group. The follicular carcinomas have larger nucleus in each cell and more monotonous chromatin pattern among cells in each case than those of the benign group. Morphological abstraction by morphometry with factor analysis may provide a practical approach to the detection of the underlying characteristics of nuclear morphology in aspiration biopsy cytology.

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Section: Discussionmentioning

confidence: 99%

Morphological abstraction of thyroid tumor cell nuclei using morphometry with factor analysis

Murata

Mochizuki

Nakazawa

et al. 2003

Microscopy Res & Technique

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“…The majority of these microscopic fluorescence studies have used the steady-state fluorescence approach with a single probe. The structural and topological changes of the DNA were interpreted in terms of changes in fluorescence intensity, which is known to be related to local DNA concentration (Bruno et al 1991; Murata 1991; Urata et al 1991; Colomb and Martin 1992; Santisteban and Brugal 1995). A few reports have focused on the DNA structure in interphase nuclei, where additional information content of the fluorescence resonance energy transfer (FRET) by steady-state fluorescence measurement was exploited (Bottiroli et al 1989; Prosperi et al 1994; Szollosi et al 2002).…”

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confidence: 99%

Spatial Distribution Analysis of AT- and GC-rich Regions in Nuclei Using Corrected Fluorescence Resonance Energy Transfer

Murata

Heřman²,

Mochizuki³

et al. 2003

J Histochem Cytochem.

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(JRL) S U M M A R Y We employed microscopic intensity-based fluorescence resonance energy transfer (FRET) images with correction by donor and acceptor concentrations to obtain unbiased maps of spatial distribution of the AT-and GC-rich DNA regions in nuclei. FRET images of 137 bovine aortic endothelial cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and corrected for the donor and acceptor concentrations by the Gordon's method based on the three fluorescence filter sets. The corrected FRET images were quantitatively analyzed by texture analysis to correlate the spatial distribution of the AT-and GC-rich DNA regions with different phases of the cell cycle. Both visual observation and quantitative texture analysis revealed an increased number and size of the low FRET efficiency centers for cells in the G 2 /Mphases, compared to the G 1 -phase cells. We have detected cell cycle-dependent changes of the spatial organization and separation of the AT-and GC-rich DNA regions. Using the corrected FRET (cFRET) technique, we were able to detect early DNA separation stages in late interphase nuclei. C ytological diagnosis of human tumors has recently become a more important clinical examination to differentiate between malignant and benign lesions. Chromatin patterns of tumor cell nuclei are among the most important information sources for cytological diagnosis. Previously we have reported a medical diagnosis based on the investigation of the chromatin structure using fluorescent DNA staining (Ashihara et al. 1986;Murata et al. 1988Murata et al. ,1990. A similar method using fluorescent DNA staining has been reported for DNA structure and cell cycle analysis, and the medical diagnosis (Latt 1974;Rousselle et al. 1999). The majority of these microscopic fluorescence studies have used the steady-state fluorescence approach with a single probe. The structural and topological changes of the DNA were interpreted in terms of changes in fluorescence intensity, which is known to be related to local DNA concentration (Bruno et al. 1991;Urata et al. 1991;Colomb and Martin 1992;Santisteban and Brugal 1995). A few reports have focused on the DNA structure in interphase nuclei, where additional information content of the fluorescence resonance energy transfer (FRET) by steady-state fluorescence measurement was exploited (Bottiroli et al. 1989;Prosperi et al. 1994;Szollosi et al. 2002).FRET is a non-radiative transfer of the excited-state energy from the initially excited donor to an excitable acceptor (Forster 1948;Stryer and Haugland 1967;Stryer 1978;Jovin and Arndt-Jovin 1989;Clegg 1992;Lakowicz 1999;Murata et al. 2000b). Because the efficiency of FRET depends on the sixth power of the distance between the donor and the acceptor, the microscopic FRET is an excellent ruler for quantification of distances on the molecular scale in cells (Jovin and Arndt-Jovin 1989).Recently we have applied fluorescence lifetime imaging microscopy (FLIM) for FRET measurements be-

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“…Since these early studies, many dyes have been shown to bind to DNA and to display increased emission (1–4). The strong emission of dyes bound to DNA also resulted in the use of fluorescence to study DNA dynamics (5, 6), chromatin structure (7, 8), and cell cycle analysis (9, 10). Specific dyes, such as Hoechst 33258 (Ho; AT specific), 4',6‐diamidino‐2‐phenylindole (AT specific), mithramycin (GC specific), and 7‐aminoactinomycin D (7‐AAD; GC specific), were used for structural analyses of AT‐ or GC‐rich DNA (11–16).…”

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Fluorescence lifetime imaging of nuclear DNA: Effect of fluorescence resonance energy transfer

et al. 2000

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Background DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady‐state measurements and do not take advantage of the additional information content of the time‐resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time‐resolved measurements to examine the proximity of donors and acceptors bound to chromatin. Methods We used frequency‐domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA‐bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT‐specific donor, Hoechst 33258 (Ho), and a GC‐specific acceptor, 7‐aminoactinomycin D (7‐AAD). Results The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT‐rich DNA throughout the nuclei. The lifetime imaging of the Ho‐stained nuclei was typically flat. Addition of 7‐AAD decreased the fluorescence intensity and lifetime of the Ho‐stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7‐AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7‐AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G2/M than in G0/1 phase cells. Conclusions Because RET efficiency depends on the average distance between Ho and 7‐AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei. Cytometry 41:178–185, 2000 © 2000 Wiley‐Liss, Inc.

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From cytofluorometry to fluorescence image analysis.

Cited by 5 publications

References 9 publications

Morphological abstraction of thyroid tumor cell nuclei using morphometry with factor analysis

Morphological abstraction of thyroid tumor cell nuclei using morphometry with factor analysis

Spatial Distribution Analysis of AT- and GC-rich Regions in Nuclei Using Corrected Fluorescence Resonance Energy Transfer

Fluorescence lifetime imaging of nuclear DNA: Effect of fluorescence resonance energy transfer

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