ARTICLE |
Correspondence to: Joseph R. Lakowicz, Center for Fluorescence Microscopy, Dept. of Biochemistry and Molecular Biology, U. of Maryland at Baltimore, Schl. of Medicine, 725 W. Lombard St., Baltimore, MD 21201.
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Summary |
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We used intensity and fluorescence lifetime microscopy (FLIM) of 3T3 nuclei to investigate the existence of AT-rich and GC-rich regions of the nuclear DNA. Hoechst 33258 (Ho) and 7-aminoactinomycin D (7-AAD) were used as fluorescence probes specific for AT and GC base pairs, respectively. YOYO-1 (Yo) was used as a dye that displays distinct fluorescence lifetimes when bound to AT or GC base pairs. We combined fluorescence imaging of Ho and 7-AAD with time-resolved measurements of Yo and took advantage of an additional information content of the time-resolved fluorescence. Because a single nucleus could not be stained and measured with all three dyes, we used texture analysis to compare the spatial distribution of AT-rich and GC-rich DNA in 100 nuclei in different phases of the cell cycle. The fluorescence intensity-based analysis of Ho- or 7-AAD-stained images indicates increased number and larger size of the DNA condensation centers in the G2/M-phases compared to G0/1-phases. The lifetime-based study of Yo-stained images suggests spatial separation of the AT- or GC-rich DNA regions in the G2/M-phase. Texture analysis of fluorescence intensity and lifetime images was used to quantitatively study the spatial change of condensation and separation of AT- and GC-rich DNA during the cell cycle.
(J Histochem Cytochem 49:14431451, 2001)
Key Words: fluorescence lifetime imaging, microscopy, texture analysis, Hoechst 33258, 7-aminoactinomycin D, YOYO-1, cell cycle
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Introduction |
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FLUORESCENCE PROBES are widely used for visualization of DNA (
In this study we combined steady-state fluorescence imaging of the nuclear DNA with FLIM to take advantage of the information content of the time-resolved fluorescence. Specifically, we used Hoechst 33258 (Ho) and 7-aminoactinomycin (7-AAD) as AT- and GC-specific stains, respectively (
One approach to looking for AT- or GC-rich regions of DNA would be to examine the same nuclei stained with each of the three dyes. For such an experiment one expects the relative brightness of 7-AAD and relative lifetime of Yo to spatially coincide and locally bright regions of Ho-stained cells to coincide with relative minima of the Yo lifetimes. However, it was not feasible using our methodology to label the nuclei with the three probes and image the intensities and lifetime of each probe. Therefore, we adopted an alternative approach. We used texture analysis (
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Materials and Methods |
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Cells and Culture Conditions
Mouse fibroblasts (3T3 Swiss albino) were grown at 37C in glass-bottomed dishes (Mat Tek; Ashland, MA) containing Dulbecco's modified Eagles's medium with 10% calf serum.
Fixation and Cell Staining
The cells in the dishes were fixed in 70% ethanol (4C for at least 30 min). After rinsing, the cells were stained with 0.4 mM Ho, 10 mM 7-AAD, or 0.5 mM Yo. All the experiments were carried out at room temperature in 10 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl. All these probes were obtained from Molecular Probes (Eugene, Oregon).
Measurement of Fluorescence Intensity and Lifetime Imaging
All fluorescence imaging experiments were done on a homodyne frequency-domain instrument based on the Zeiss Axiovert 135TV inverted fluorescence microscope. The Ho fluorophore was excited at 335 nm by a frequency-doubled output from the synchronously-pumped DCM dye laser. The Yo and 7-AAD were excited by an output of the mode-locked Ar+ laser at 514.5 nm. The pulsed laser excitation was directed to the microscope by a quartz optical fiber which was continuously shaken during data acquisition to randomize the excitation light field. The microscope worked in the epi-illumination mode and was equipped with the Zeiss C-Apochromat x40/1.2 water immersion objective and a x1.6 Optovar insert. The Zeiss FT 395, Omega 515DLRP, and Omega 595 DRLP dichroic mirrors were used for Ho, Yo, and 7-AAD, respectively. Fluorescence from the sample was isolated by broad-band interference filters Omega 450DF65, Intor 550/40, and Omega 635DF55 for Ho, Yo, and 7-AAD, respectively. The sample emission was directed to the Hamamatsu C5825 high-speed image intensifier and registered by the Photometrics PXL scientific-grade, slow-scan, cooled CCD camera. The correct focus was adjusted on the nuclear margins. For time-resolved experiments with Yo, the gain of the image intensifier was modulated at 75.468 MHz using an output of the PTS 300 synthesizer phase-locked to the master oscillator of the pumping Ar+ laser mode locker. Eight images, with the detector phase equally spaced over 360°, were acquired for each lifetime measurement. Data were analyzed by a method described elsewhere (
Cell Cycle Analysis
The fluorescence intensity of each nucleus is related to the amount of DNA in the nucleus. We measured the total fluorescence intensity emitted from the nuclei and constructed DNA histograms for the cell cycle analysis.
Texture Analysis of Fluorescence Intensity and Lifetime Imaging
The fluorescence intensity images of 100 cells stained with Ho and 7-AAD and lifetime images of 100 cells stained by Yo were measured. A texture of the images was characterized by texture parameters that were calculated from co-occurrence matrices P(i,j) (
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The terms Px+y(k) and Px-y(k) are defined as follows:
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(4) |
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(5) |
where P(i,j) is the (i,j)-th entry in a normalized gray-tone spatial-dependency matrix (co-occurrence matrix) and Ng is the number of distinct gray levels in the image. The parameters (ASM, SVar and DVar) were calculated for every nuclear image from the ensemble of 100 stained cells. In particular, we calculated mean value of the texture parameter by averaging values obtained from four angular co-occurrence matrices constructed for angles 0, 45, 90, and 135°. A one-pixel correlation distance was used for construction of the matrices.
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Results |
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Three different fluorescent DNA probes were used to study cell cycle-dependent changes of the DNA distribution pattern in nuclear DNA of 3T3 fibroblasts. The Ho and 7-AAD were chosen for their ability to specifically bind and strongly fluoresce in the AT- and GC-rich regions of DNA, respectively (
Fluorescence Intensity Imaging of Ho and 7-AAD
Fig 1 shows a histogram of integrated fluorescence intensity originating from the cell nucleus for an ensemble of 100 cells. The histogram clearly shows two distinct peaks. Because the total fluorescence intensity is proportional to the DNA content, which varies during the cell cycle, the peaks can be assigned to cells in the particular stage of the cell cycle. For simplicity, the peak with the lowest intensity belongs to the cells in the G0/1-phase, which possess a single set of chromospheres. The second peak, with approximately doubled fluorescence intensity, belongs to the cells in the G2/M-phase, which contain double the amount of DNA. Cells with even higher fluorescence intensity are anomalous polyploid cells. Having constructed the histogram from Fig 1, we can assign to the each cell a position within the cell cycle. From the shape of the histogram we can also conclude that Ho is more suitable for the cell cycle analysis than 7-AAD, as it gives better separation of the intensity peaks.
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An example of the intensity images of cells stained by Ho or 7-AAD is shown in Fig 2. The Ho-stained images exhibit higher heterogeneity and a higher number of bright spots compared to the images of the 7-AAD-stained cells (
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Comparison of the images from Fig 2 reveals that the nuclear area of the cells in the G2/M-phase is almost two times larger than that in the G0/1-phase. In addition, the number of bright spots that represent AT- or GC-rich regions is increased in the G2/M-phase compared to the G0/1-phase. The same morphological changes can be traced in histograms and co-occurrence matrices of those images, Fig 3 and Fig 4, respectively. In particular, the co-occurrence matrices of the normalized images from Fig 2 show different gray-scale amplitudes (Fig 4). This can be seen from the length of the pattern aligned along the diagonal of the matrix. The Ho-stained nucleus shows a higher amplitude of the intensity profile compared to the 7-AAD-stained nucleus. Assuming smooth intensity change, this observation suggests that the AT-rich DNA is more localized than the GC-rich DNA. An analysis of the patterns in the perpendicular direction (parallel to the other diagonal) can give a qualitative estimate of the image contrast. The broader the distribution in this direction, the higher contrast the image has. Visually, there is a smaller contrast in the images in the G2/M-phase (Fig 4). This finding is confirmed by the texture analysis of the whole ensemble of cells. Fig 5 shows results of the cell cycle-related texture analysis of 100 fluorescence intensity images of nuclei stained separately by Ho and 7-AAD. For both dyes, the ASM value is higher for nuclei in the G0/1-phase compared to the nuclei in the G2/M-phase. Because the increased value of ASM indicates higher homogeneity of the image, the result suggests more homogeneous spatial distribution of the DNA in the G1/0-phase. The decreased homogeneity in the G2/M-phase arises from the increased size and number of the bright spots. In particular, both AT- and GC-rich DNA regions seem to become more condensed and localized in the G2/M-phase (
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The SVar parameter, describing the distribution pattern along the upper left lower right diagonal of the co-occurrence matrix shows no dependence on the cell cycle. The result suggests that the overall amplitude of the intensity changes does not depend on the cell cycle.
The DVar values, characterizing mean contrast of the image, show that nuclei in the G2/M-phase exhibit comparatively lower contrast than nuclei in the G0/1-phase. The DVar decreases with a decreasing contrast and is related to the steepness of the intensity changes. Because the amplitude of the intensity changes stays constant, the decreased contrast means that the intensity changes take place over a greater distance. In other words, constant value of SVar and decreased values of DVar indicate larger size of the condensed DNA regions in the G2/M-phase.
Fluorescence Lifetime Imaging of Yo
The spatial distribution of the AT- and GC-rich regions of the YO-stained nuclear DNA was studied by the lifetime imaging. Yo is a nonspecific DNA dye with a lifetime sensitive to the local environment. In an in vivo study it was observed that the fluorescence decay of Yo in poly[dG-dC] is close to a single exponential with a mean lifetime of 4.12 nsec. In the poly[dA-dT] the mean lifetime shortens to 1.48 nsec and fluorescence decay becomes highly heterogeneous (
Fig 6 shows the modulation and the phase lifetime image of the Yo-stained nucleus. As expected, both images exhibit significant spatial variations of lifetimes. The lower panel shows correlation between modulation and phase lifetime on a pixel-to-pixel basis. The lifetime image of the nucleus contains three classes of regions. In the first class, the values of the phase and modulation lifetimes are close to each other (Fig 6A6C, Region a). This is an indication of the mono-exponential intensity decay and of the high GC:AT ratio. Region b is characterized by both lifetimes being shorter. Moreover, the phase lifetime is substantially shorter than the modulation lifetime. This is an indicator of fluorescence decay heterogeneity. Such decays are expected to be measured in regions containing excess of AT base pairs. The fluorescence decay in the third region (Region c) is heterogeneous with lifetimes close to the values found for nonspecific DNA (
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Fig 7 shows cell cycle analysis of the ensemble of 100 Yo-stained cells. Cells were indexed and sorted according to their total fluorescence intensity and position within the cell cycle. Fig 8 and Fig 9 show an example of modulation and phase lifetime images of randomly chosen cells in the G0/1 and G2/M-phases and corresponding pixel-based lifetime histograms. The co-occurrence matrices of the images from Fig 8 are presented in Fig 10. All lifetime images display spatial heterogeneity of lifetimes. The phase lifetime images of cells in the G2/M-phase exhibit larger size and larger number of the low-lifetime dark spots, which is a indication of an excess of AT base-pairs (Fig 8c and Fig 8d). This effect appears to be weaker in the modulation lifetime images (Fig 8a and Fig 8b). Histograms presented in Fig 9 display broad unimodal lifetime distributions centered near 2.2 nsec and 1.9 nsec for modulation and phase lifetime, respectively. However, the co-occurrence matrices of both the phase and modulation lifetime images from Fig 8 show similar characteristics. In particular, the size of the pattern aligned along the top left to the bottom right diagonal reveals slightly larger amplitude of the lifetime variation in the G2/M cell compared to the G0/1 cell (Fig 10). This single cell-based observation was confirmed by the statistical analysis of 100 cells, which shows increase of the SVar value in the G2/M-phase (Fig 11).
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Fig 11 shows cell cycle-dependent statistical analysis of the co-occurrence matrices for all 100 cells. Whereas values of ASM and DVar show no statistically significant dependence on the cell cycle, the value of SVar becomes higher in the G2/M-phase. For a constant value of DVar, the value of SVar should increase with the size of the domains. Our result indicates spatial separation of the AT- and GC-rich DNA regions in the G2/M cells. Invariance of ASM and DVar is consistent with the observed independence of the mean lifetime on the cell cycle (Fig 12). This result is in accord with the knowledge that the overall amount of nuclear DNA changes during the cell cycle but that the global AT:GC base pair ratio remains constant.
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Discussion |
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In this work we studied cell cycle-associated spatial changes of AT- and GC-rich regions in an ensemble of cells by texture analysis. We considered the structural changes of DNA in terms of the relative spatial separation as well as the condensation of the AT- and GC-rich regions in the nuclei. Texture analysis of the fluorescence intensity images gives informative data on the DNA condensation in nuclei, as was reported by a number of groups (
Nuclei stained by AT-specific Ho and GC-specific 7-AAD reveal bright fluorescence in locations with a high concentration of DNA. The texture analysis of the intensity images indicates that, compared to the G0/1-phase, in the G2/M-phase the DNA concentration becomes spatially more heterogeneous with increasing number and size of the spatially well-defined condensation centers.
The fluorescence lifetime images are independent of the local DNA concentration (
In conclusion, the steady-state fluorescence intensity-based texture analysis shows condensation of the AT- and GC-rich DNA. Lifetime images reveal a spatial relation between AT- and GC-rich regions. Both steady-state fluorescence intensity and the lifetime-based texture analysis reveal significant rearrangement of the nuclear DNA during the cell cycle.
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Footnotes |
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1 On leave from First Department of Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
2 On leave from Institute of Physics, Charles University, Prague, Czech Republic.
Received for publication October 23, 2000; accepted May 2, 2001.
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