Texture Analysis of Fluorescence Lifetime Images of AT- and GC-rich Regions in Nuclei

Murata, Shin‐ichi; Heřman, Petr; Lakowicz, Joseph R.

doi:10.1177/002215540104901112

Cited by 55 publications

(43 citation statements)

References 22 publications

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“…Despite the same staining conditions for all cells on the dish, both donor and acceptor concentrations in every nucleus exhibit a spatial heterogeneity resulting from a non-uniform AT and GC base-pair distribution, respectively (Murata et al 2001a). The high local AT and GC concentrations, observed as bright spots, could be caused either by the presence of AT-and GC-rich segments of DNA, respectively, or it could reflect higher base-pair concentrations caused by local condensation of the nonspecific DNA.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…We have studied the spatial relation between regions of the AT-and GC-rich DNA (Murata et al 2000a(Murata et al ,2001a. The fluorescence lifetime measurements of the donor in the presence of the acceptor revealed a higher FRET efficiency in the AT-rich DNA regions compared to the GC-rich regions (Murata et al 2000a).…”

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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: Discussionmentioning

confidence: 99%

Section: Fluorescence Intensity and Fret Measurementsmentioning

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.

Self Cite

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“…The applications of FLIM in the biosciences are manifold and reach from monitoring cellular parameters (Ca 2+ 55-57 and Na + concentrations 58 , pH [59][60][61][62][63][64] , pO 2 and ROS concentrations [65][66][67] , pCO 2 68 ) and the distances between proteins on the nm-scale by means of FRET 5,35,42,[46][47][48][68][69][70][71][72][73][74][75][76][77][78][79][80][81] to observing central cellular processes in real time, e.g. interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] .…”

Section: Bioscientific Applicationsmentioning

confidence: 99%

Fluorescence lifetime imaging in biosciences: technologies and applications

Niesner

Gericke

2008

Front. Phys. China

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The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.

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“…Dedicated instruments that use the frequency-domain approach are available (e.g., Lambert Instruments, Leutingewolde, The Netherlands) (Verveer et al, 2000(Verveer et al, , 2001. In the time domain approach, pulsed or pulse-modulated laser excitation or gated bright LEDs are used as the excitation source (Chen and Periasamy, 2004;Elangovan et al, 2002;Murata et al, 2001). A fast photomultiplier is used to derive the lifetime by accumulating a histogram of the intervals between pulse generation and photon emission.…”

Section: Fluorescence Lifetime Imagingmentioning

confidence: 99%

Studying inner ear protein–protein interactions using FRET and FLIM

et al. 2006

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Molecular genetic studies of the inner ear have recently revealed a large number of previously undescribed proteins, but their functions remain unclear. Optical methods such as FRET and FLIM are just beginning to be applied to the study of functional interactions between novel inner ear proteins. This review discusses the various methods for employing FRET and FLIM in proteinprotein interaction studies, their advantages and pitfalls, with examples drawn from inner ear studies.

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Texture Analysis of Fluorescence Lifetime Images of AT- and GC-rich Regions in Nuclei

Cited by 55 publications

References 22 publications

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

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

Fluorescence lifetime imaging in biosciences: technologies and applications

Studying inner ear protein–protein interactions using FRET and FLIM

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