Multiparametric analysis using autostage cytofluorometry.

Ashihara, Tsukasa; Kamachi, M.; Urata, Yoji; Kusuzaki, Katsuyuki; Takeshita, Hideyuki; Kagawa, Keiichiro

doi:10.1267/ahc.19.51

Cited by 24 publications

(9 citation statements)

References 11 publications

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“…With the acceptor filter set we measured the total acceptor fluorescence from 137 double-stained cells, evaluated the amount of DNA in the nucleus, and constructed DNA histograms for the cell cycle analysis (Ashihara et al 1986;Murata et al 2000aMurata et al ,2001a. Since we worked with unsynchronized cell colonies, cells from our ensemble were randomly distributed along the cell cycle.…”

Section: Fluorescence Intensity and Fret Measurementsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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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Section: Fluorescence Intensity and Fret Measurementsmentioning

confidence: 99%

mentioning

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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show abstract

“…In the present study concerning colorectal carcinoma, we have collected both of these values sequentially on an identical nucleus by using computer controlled auto-scanning stage [1]. We have paid attention to the heterogeneity in DNA profile, and compared the number of signals in a nucleus (number of S/N) among cytofluorometrically distinct subpopulations, in order to investigate the correlation between nuclear DNA content and numerical aberration of chromosome 17.…”

Section: Recentlymentioning

confidence: 99%

The Relationship between Numerical Aberrations of Chromosome 17 and Nuclear DNA Content in Colorectal Carcinoma Detected by Fluorescent In Situ Hybridization (FISH) and Cytofluorometry Using Auto-scanning Stage.

Kawai

Hamada

Yamagishi

et al. 1997

Acta Histochem. Cytochem

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“…Since morphological observation yields important information on the cell, we have been studying the methods of microscope-based cytofluorometry for multiparametric analysis (1)(2)(3)10). In this article, we describe these methods of quantitative cytological analysis based on studies from cytofluorometry to image analysis.…”

mentioning

confidence: 99%

“…Such a method promises wide application in cytofluorometry, such as 1) cytofluorometry in combination with observation under transmitted light after conventional non-fluorescence staining, or with autoradiography without optical interference, 2) two parametric cytofluorometry, including the analysis of cell samples prepared with fluorescence dyes with similar fluorescence spectra, and 3) multiparametric cytofluorometry with more than two fluorescence stainings. The following system, which has an autoscanning stage (autostage), has been developed in our laboratory to accomplish these tasks, based on the prototype previously reported (2). Autostaging Cytofluorometry Fig.…”

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confidence: 99%