ARTICLE |
Correspondence to: Shin-ichi Murata, Dept. of Pathology, U. of Yamanashi School of Medicine, 1110 Shimokato, Tamaho-cho Nakakoma-gun, Yamanashi, Japan, 409-3898. E-mail: smurata@res.yamanashi-med.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 G2/M-phases, compared to the G1-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. (J Histochem Cytochem 51:951958, 2003)
Key Words: corrected FRET, fluorescence resonance energy, transfer, texture analysis, DNA, Hoechst 33258, 7-aminoactinomycin D, cell cycle
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CYTOLOGICAL 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 (
FRET is a non-radiative transfer of the excited-state energy from the initially excited donor to an excitable acceptor (
Recently we have applied fluorescence lifetime imaging microscopy (FLIM) for FRET measurements between the AT-specific dye Hoechst 33258 (Ho) and the GC-specific dye 7-aminoactinomycin D (7-AAD) bound in cell nuclei (
The efficiency of FRET in the donoracceptor system with multiple acceptors is known to depend on the acceptor concentration. Although sensitive to FRET, donor fluorescence lifetimes are independent of the local donor concentration (
In this study we decided to extend our previous FLIM work and describe in more detail cell cycle-related changes of the DNA organization, while taking into account highly variable donor and acceptor concentrations in the nucleus. We have measured energy transfer between Ho and 7-AAD by steady-state fluorescence microscopy, and differences of the FRET signal before and after the Gordon's correction method were compared. Images of the cell cycle-dependent FRET signal were classified by texture analysis (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells and Fluorescence Staining
Bovine aortic endothelial cells were grown at 37C in 35-mm Falcon dishes (Becton Dickinson; Franklin Lakes, NJ) containing DME with 10% FCS. The unsynchronized cell colonies in the dishes were fixed in 70% ethanol (4C, at least 30 min). After a rinse, the cells were stained with 0.8 µM Ho and 1.6 µM 7-AAD, which were used as the donor and the acceptor, respectively. The concentrations of donor and acceptor were determined to be able to measure significant changes in FRET signal. The concentration of 7-AAD typically resulted in about a twofold decrease in the Ho fluorescence intensity. All experiments were carried out at room temperature in 10 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl (
Image Acquisition
Images of the stained cell cultures were acquired using an epi-illumination fluorescence microscope BX50 (Olympus; Tokyo, Japan) with an UplanApo x40 oil immersion objective (NA 1.0). The DNA fluorescence was recorded by a scientific-grade cooled CCD camera, Sensys (Photometrics; Tucson, AZ) connected to a PC. The images were pre-processed by IPLab software (Scanalytics; Fairfax, VA). The illumination level of the sample was adjusted by neutral density filters to the level that the image acquisition time was in the order of seconds. No detectable photobleaching was observed under these conditions.
Fluorescence Intensity and FRET Measurements
Fluorescence intensity images of 137 cells on a single dish, double-stained by Ho and 7-AAD, were measured. Three different filter sets were used for the FRET measurement: (a) the donor filter set consisting of a 360370-nm excitation filter, a 400-nm dichroic mirror, and a 420460-nm emission filter; (b) the acceptor filter set consisting of a 530550-nm excitation filter, a 590-nm dichroic mirror, and a 590-nm longpass emission filter; and (c) the FRET filter set consisting of a 360370-nm excitation filter, a 400-nm dichroic mirror, and a 590-nm longpass emission filter. With a complete absence of non-FRET signals, a simple FRET measurement can be done by observing the acceptor emission while exciting the donor. Since the non-FRET signals cannot be completely eliminated, the FRET signal IFRET can be calculated from the following equation (
where F(da) is a fluorescence signal from the cell in the presence of both donor and acceptor collected with the FRET filter set, D(da) is a signal in the presence of both donor and acceptor collected with the donor filter set, A(da) is a signal in the presence of both donor and acceptor using the acceptor filter set, F(d) is a signal from a donor-only stained cell using the FRET filter set, D(d) is a signal from a donor-only stained cell with the donor filter set, F(a) is a signal from the acceptor-only stained cell acquired with the FRET filter set, and A(a) is a signal from the acceptor-only stained cell using the acceptor filter set. The constant ratios F(d)/D(d) and F(a)/A(a) depend on the fluorophores and on the instrumentation.
The fluorescence intensity IFRET calculated from Eq. 1 is subtracted from the non-FRET signals but is not corrected for the concentration of the donor and acceptor. Corrected FRET signal IcFRET, where the bias caused by the concentration of donor and acceptor as well as by the fluorescence crosstalk is removed, can be calculated from the following equation (
The constant factor G relates a FRET-induced loss of the donor emission intensity observed with the donor filter set to the gain of the acceptor fluorescence intensity observed with the FRET filter set (
Because the donor cannot be excited with the acceptor filter set, the overall signal from the double-stained nucleus contained fluorescence from the acceptor only and was related to the amount of the DNA in the nucleus. 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 (
Texture Analysis of Co-occurrence Matrices
The high-dimensional texture analysis of the co-occurrence matrices is a classical method sensitive enough to detect subtle changes in the DNA organization (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our donoracceptor pair exhibits a good spectral match for the FRET experiments. Spectral overlap of the absorption and the emission spectra of the Ho (the AT-specific dye) and 7-AAD (the GC-specific dye), respectively, is shown in Fig 1. Using this pair of the fluorochromes, we examined FRET in the nuclei of 137 bovine aortal endothelial cells. The nuclear images typically displayed spatial heterogeneity of the fluorescence intensity with a number of brighter spots (Fig 2A and Fig 2B). We found differences in the distribution and size of the bright spots between the Ho-stained and the 7-AAD-stained cells. As previously found, the Ho-stained images exhibited higher heterogeneity and a greater number of bright spots compared to the images of the 7-AAD-stained cells (
|
|
Fig 2C and Fig 2D show the FRET images before and after the three-filter-set correction, respectively. Eq. 1 separates the FRET signal from the non-FRET signals, but is not normalized for the concentration of the donor and acceptor. On the other hand, the corrected FRET image (IcFRET image) calculated from Eq. 2 was corrected for both non-FRET signal and the donor and acceptor concentrations. As expected, different patterns were observed before and after the correction. Both FRET images from Fig 2C and Fig 2D exhibited considerable spatial heterogeneity. Regions of high FRET signal in the uncorrected FRET image were correlated with regions of low values in the corrected FRET image. These apparently opposite results strongly stress the need for concentration corrections, since high local concentration of the donoracceptor pairs can completely offset and even dilute the loss of the intensity-based FRET signal. Interestingly, the lowest corrected energy transfer was found in regions with the highest DNA concentration (Fig 2A, Fig 2B, and Fig 2D), indicating higher donoracceptor distances and consequently larger separation of AT- and GC-rich DNA segments in these locations.
Fig 3A shows a histogram of the integrated fluorescence intensities collected from the nuclei of 137 cells. Because the integrated intensity is proportional to the DNA content, we could assign to each cell a position within the cell cycle. Having the histogram from Fig 3A, we compared the IcFRET images of cells from different stages. Fig 3B3Q show 16 representative IcFRET images of cells in the G1- and S-G2/M-phases. Regions with a low FRET signal are shown as light and dark blue spots. Visually, the size of the blue regions appeared to be larger in the G2/M-phases compared to the G1-phase. The value of the IcFRET was lower in the G2/M-phases than in the G1-phase, as shown by a comparison of the mean IcFRET values calculated from whole nuclear areas (Fig 4). As seen in Fig 4, cells in the G2/M-phases exhibited about 40% lower cFRET signal than cells in the G1-phase.
|
|
To confirm the observation demonstrated in Fig 3, we employed methods of the texture analysis (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As reported by number of groups, the fluorescence intensity images provide informative data on the DNA condensation in nuclei (
FRET in this study was measured between dyes bound to the double-helical DNA. In this case, multiple acceptors are distributed around donors on DNA, and the extent of FRET depends on donor and acceptor concentrations (
Previously we demonstrated that addition of 7-AAD to Ho-stained nuclei results in a shortening of the donor fluorescence lifetime (
During the cell cycle, three-dimensional distribution of chromosomes changes in nuclei. Finally, the AT- and GC-rich DNA regions of each chromosome separate in the metaphase (
In conclusion, we have demonstrated that corrected FRET imaging with texture analysis is a useful tool for analysis of the intracellular DNA organization and gives information inaccessible by non-FRET steady-state fluorescence microscopy. The corrected FRET technique can be applied as a helpful tool that will give us additional information about the chromatin pattern for medical cytological diagnosis of human tumors.
![]() |
Acknowledgments |
---|
Supported by the NIH, National Center for Research Resources, RR-08119.
We thank our colleagues Dr. T. Yamane and Dr. A. Ooi for the preparation of cell cultures and for the technical support of the microscopic and imaging equipment.
Received for publication August 12, 2002; accepted February 4, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular Biology of the Cell. 3rd ed New York, London, Garland Publishing
Ashihara T, Kamachi M, Urata Y, Kusuzaki K, Takeshita H, Kagawa K (1986) Multiparametric analysis using autostage cytofluorometry. Acta Histochem Cytochem 19:51-59
Bottiroli G, Croce AC, Gerzeli G, Barni S (1989) DNA double staining for a fluorescence energy transfer study of chromatin in liver cells. Cell Biophys 15:249-263[Medline]
Bruno S, Crissman HA, Bauer KD, Darzynkiewicz Z (1991) Changes in cell nuclei during S phase: progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34. Exp Cell Res 196:99-106[Medline]
Clegg R (1992) Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol 211:353-389[Medline]
Colomb E, Martin PM (1992) S phase, an evolutionary chromatin condensation state from G1 to G2, in a breast epithelial cell line. Anal Cell Pathol 4:369-379[Medline]
Forster T (1948) Intermolecular energy migration and fluorescence (translated by R. S. Knox, Department of Physics and Astronomy, University of Rochester, Rochester, NY). Ann Phys 2:55-75
Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74:2702-2713
Haralick RM (1992) Texture. In Haralick RM, Shapiro L, eds. Computer and Robot Vision. New York, Addison Wesley, 453-507
Haralick RM, Shanmugam K, Dinstein I (1973) Texture features for image classification. IEEE Trans Systems, Man and Cybernetics SMC 3:610-621
Jovin T, ArndtJovin D (1989) FRET microscopy: digital imaging of fluorescence resonance energy transfer. Application in cell biology. 9th ed. In Kohen E, Ploem JS, Hirschberg JG, eds. Cell Structure and Function by Microspectrofluorometry. Orlando, Academic Press, 99-117
Lakowicz J (1999) Principles of Fluorescence Spectroscopy. 2nd ed New York, Kluwer Academic/Plenum Publishers
Lakowicz JR, Szmacinski H, Nowaczyk K, Berndt KW, Johnson M (1992) Fluorescence lifetime imaging. Anal Biochem 202:316-330[Medline]
Latt S (1974) Detection of DNA synthesis in interphase nuclei by fluorescence microscopy. J Cell Biol 62:546-550
Latt SA, Sahar E, Eisenhard ME (1979) Pairs of fluorescent dyes as probes of DNA and chromosomes. J Histochem Cytochem 27:65-71[Abstract]
Murata S (1991) Correlated analysis of nuclear DNA content and nuclear morphological features in the thyroid hyperplasias and tumors using image cytometry. J Kyoto Pref Univ Med 100:311-331
Murata S, Herman P, Lakowicz J (2001a) Texture analysis of fluorescence lifetime images of AT- and GC-rich regions in nuclei. J Histochem Cytochem 49:1443-1452
Murata S, Herman P, Lakowicz J (2001b) Texture analysis of fluorescence lifetime images of nuclear DNA with effect of fluorescence resonance energy transfer. Cytometry 43:94-100[Medline]
Murata S, Herman P, Lin H-J, Lakowicz J (2000a) Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer. Cytometry 41:178-185[Medline]
Murata S, Itoi H, Urata Y, Tsuchihasi Y, Matsuzuka F, Ashihara T (1990) A combined, quantitative analysis of DNA content and nuclear morphology of thyroid follicular tumor using an autostaging cytofluorometer combined with an image processor. Jpn Soc Clin Cytol 29:819-828
Murata S, Kubo H, Itoi H, Konishi E, Urata Y, Ashihara T (1993a) Basic technique of image analysis for quantitative histochemistry and cytochemistry. In Histochemistry and Cytochemistry 1993. Tokyo, Japanese Society of Histochemistry and Cytochemistry, Gakusai-kikaku, 126139
Murata S, Kubo H, Urata Y, Ashihara T (1993b) Preliminary study on texture analysis for cell nuclear morphology. Acta Histochem Cytochem 26:468
Murata S, Kusba J, Piszczek G, Gryczynski I, Lakowicz J (2000b) Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentration. Biopolymers 57:306-315[Medline]
Murata S, Nakamura N, Nakazawa T, Kondo T, Mochizuki K, Yamashita H, Urata Y et al. (2002) Detection of underlying characteristics of nuclear chromatin patterns of thyroid tumor cells using texture and factor analyses. Cytometry 49:91-95[Medline]
Murata S, Urata Y, Shima M, Ozaki T, Matsui M, Tokuda H, Ashihara T (1988) A case report of malignant insulinoma: immunohistochemical and DNA-cytofluorometric studies. Pathol Clin Med 6:1095-1100
Prosperi E, Giangare MC, Bottiroli G (1994) DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ. Histochemistry 102:123-128[Medline]
Rousselle C, Paillasson S, RobertNicoud M, Ronot X (1999) Chromatin texture analysis in living cells. Histochem J 31:63-70[Medline]
Santisteban MS, Brugal G (1995) Fluorescence image analysis of the MCF-7 cycle related changes in chromatin texture. Differences between AT- and GC-rich chromatin. Anal Cell Pathol 9:13-28[Medline]
Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819-846[Medline]
Stryer L, Haugland R (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 58:719-726[Medline]
Szollosi J, Damjanovich S, Mulhern SA, Tron L (1987) Fluorescence energy transfer and membrane potential measurements monitor dynamic properties of cell membranes: a critical review. Prog Biophys Mol Biol 49:65-87[Medline]
Szollosi J, Nagy P, Sebestyen Z, Damjanovicha S, Park J, Matyus L (2002) Applications of fluorescence resonance energy transfer for mapping biological membranes. J Biotechnol 82:251-266[Medline]
Urata Y, Itoi H, Murata S, Konishi E, Ueda K, Azumi Y, Ashihara T (1991) From cytofluorometry to fluorescence image analysis. Acta Histochem Cytochem 24:367-374