ARTICLE |
Correspondence to: Zvi Malik, Life Sciences Dept., Bar Ilan University, Ramat-Gan 52900, Israel.
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Summary |
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Chromatin condensation and nuclear organization of May-Grunwald-Giemsa (MGG)-stained normal erythropoietic bone marrow cells and apoptotic red cell precursors were resolved by spectral bio-imaging. Multipixel spectra were obtained from single cells displaying a range of wavelengths of both transmitted and absorbed light. Two groups of spectra, of low- and high-intensity transmitted light, were revealed in the nuclei of each cell. The absorbance spectra served for the reconstruction of "absorbance images" depicting the affinity of MGG stain for the chromatin of proerythroblasts and of basophilic, polychromatic, and orthochromatic normoblasts. The localization of different spectral components in the nuclei was resolved employing two mathematical methods, spectral similarity mapping and principal component analysis. Novel structures of high symmetry revealing windmill-like organization were detected in basophilic, polychromatic, and orthochromatic normoblast cells. Matching structures were detected in apoptotic normoblasts obtained from an agnogenic myeloid metaplasia patient. Apoptosis was associated with a gradual breakdown of the ordered arrays in the nucleus. We propose that DNA cleavage may lead to fragmentation of the symmetrical windmill-like superstructure of the basic nuclear domains. (J Histochem Cytochem 45:1097-1108, 1997)
Key Words: multipixel spectroscopy, spectral similarity mapping, absorbance image, eigen images, apoptosis imaging, erythropoiesis imaging
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Introduction |
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Terminal erythroid differentiation and apoptosis share common processes of nuclear chromatin condensation and cell volume decrease. However, morphological and biochemical distinctions between these two processes have been described (
Nuclear chromatin structure is modulated by histone-histone, histone-DNA interactions, histone modifications, and by the presence of non-histone proteins (
During maturation of the red cell precursors, the nucleus exhibits a series of qualitative morphological changes (
Apoptosis is a process of active cell death associated with distinctive morphological and biological events such as nuclear condensation (pyknosis) and fragmentation (karyorrhexis), with internucleosomal cleavage of cellular DNA (
The hematopoietic progenitor cells in folate-deficient individuals have nuclear irregularities including increased size, lobulation, and fragmentation. These abnormalities in the nuclei and the DNA imply the disruption of biochemical reactions that require folate co-enzymes and are in a pathway that lead to DNA synthesis (
Biological applications of image analysis include the analysis of chromosomal structure, cell ploidity quantitative immunohistochemistry (
Standard analysis of blood cells for the determination of differentiation and pathological conditions is based on staining with MGG or Romanowsky techniques, which employ the dyes azure B and eosin. Spectroscopic selected area microanalysis has been shown to enhance the data obtained from cells stained by standard methods. By a spectral subtraction technique,
We have recently described new techniques of Fourier transform multipixel spectroscopy for light microscopy (
To determine chromatin organization in erythroid cells stained with MGG, we employed a novel technique of spectrally resolved image analysis. Morphological patterns of chromatin organization during normal erythroid differentiation and apoptosis were determined by mathematical methods of spectral similarity mapping and principal component analysis.
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Materials and Methods |
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Fourier Transform Multipixel Spectrometry System for Microscopy
Spectral imaging was performed using the SpectraCube SD-200 (Applied Spectral Imaging; Migdal HaEmek, Israel). The SpectraCube system consists of an interferometer situated in the parallel beam between an objective lens (infinity corrected) and a lens equivalent to an eyepiece, whose purpose is to form an image on a CCD camera. The light beam passing through the specimen is split in the interferometer in opposite directions, and is united again at the exit with an optical path difference (OPD) that is a function of the angle between the incoming beam and the interferometer itself. The OPD arises because for non-zero angles the two beams undergo different optical paths in the beamsplitter. The inherent mechanical stability of this interferometer allows the Fourier technique to be successfully applied to the visible spectral region. The measurement is done by recording successive CCD frames in synchronization with the steps of the motor used to rotate the collimated beam, so that the instantaneous OPD is known for every pixel in every recorded frame and can be used in the FFT calculation (
Briefly, in spectral imaging, each pixel is actually one of several tens of thousands of microspectrometers, acting simultaneously and independently. As a result, spectral imaging acquires a so-called cube whose appellate signifies the two spatial dimensions of a flat sample (x and y), and the third wavelength dimension. The calculated pixel size in a spectral image is 0.04 µm2. The spectral resolution (FWHM, full width at half maximum) is 5 nm at 400 nm (12 nm at 600 nm) and the spectral range (more than 5% response) is 400-1000 nm (
Optical Density and Absorbance Images
The estimation of chromatin optical density was obtained by applying the Beer-Lambert law for absorbance:
Similarity Mapping Analysis
To segregate different regions in the sample, we employed the spectral similarity mapping mathematical function, which calculates the differences between area integrals of one chosen spectrum with respect to all the other spectra composing an image. The comparison algorithm used in this work for similarity mapping is defined by the following function:
The reconstructed similarity map image composed of bright and gray pixels reveals the degree of similarity between the spectra. The brighter the pixel, the more the two spectra are alike.
Principal Component Analysis
Principal component analysis (also referred to as eigenvector) uses linear transformation of multiband data to translate and rotate data into a new coordinate system that maximizes the variance. This technique is useful for enhancing the information content, segregating noise components, and for reducing the dimensionality of data sets. The original data are arranged in a population of x vectors. The components of each vector are the intensity values of a certain pixel at the different spectral slices. The covariance matrix of the vector population is defined as:
May-Grunwald Staining of Bone Marrow Specimens
Bone marrow aspiration biopsy specimens were obtained from patients with non-hematologic disorders and peripheral blood from a patient with agnogenic myeloid metaplasia (AMM) with a leukoerythroblastic blood picture (
Flow Cytometry
Apoptotic cells were assayed for DNA content using the propidium iodide (PI) staining method and subsequent flow cytometry analysis. Briefly, the cells (usually 2 x 106) were washed twice with PBS, centrifuged, and fixed in 70% ethanol. The fixed cells were pelleted, resuspended in PBS, and incubated at 37C for 30 min with 100 µg/ml RNAse A and 5 µg/ml PI [prepared in PBS at 100 µg/ml and kept in the dark at room temperature (RT)]. Analyses of the cells for cell-cycle status were performed using a Becton-Dickinson FACSort. For each sample, 10,000 cells were analyzed with gating to exclude doublets. The data were collected and deconvoluted by a cell-fit program.
Electron Microscopy
For transmission electron microscopy, the cells were fixed with 2.0% paraformaldehyde and 2.5% glutaraldehyde for 1 hr at RT. The cells were postfixed with 1% osmium tetroxide for an additional hour at 4C, dehydrated in alcohol, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and photographed using a JEOL 1200EX transmission electron microscope.
For scanning electron microscopy, the fixed cells were washed twice in Ca+2 + Mg+2-free Dulbecco's phosphate-buffered saline and dehydrated by immersion in a gradient of ethanol solutions. Then ethanol was exchanged with Freon 113 and the cells were critical point-dried. Specimens were coated with gold and micrographs were taken with a JEOL JSM 840 scanning electron microscope.
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Results |
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Spectral Analysis
Figure 1A-D show MGG-stained erythropoietic progenitor cells and their corresponding multipixel transmitted light spectra of the chromatin. Each spectrum in Figure 1E-H is the average of a sample of 10 pixels arbitrarily chosen from two domains, one with low-intensity light transmittance (LIT) and the other with high-intensity light transmittance (HIT), out of 4 x 104 pixel spectra composing multiple cellular sites of the single cell in Figure 1A-D. Therefore, the spectrally resolved image of an erythropoietic cell is a three-dimensional set of data: f (x,y,) for each pixel at plane x,y; the third dimension is its wavelength
. The transmittance spectra of progenitor erythroid cell nuclei depict a similar pattern: a low-intensity light transmittance region in the range of 550-750 nm in comparison to the high-intensity light transmittance region. Therefore, the spectra of corresponding cells in Figure 1A-D may show some specific changes related to the differentiation and cytological transformation, as shown in Figure 1E-H.
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Figure 1I-L show a complementary data set of spectra revealing the chromatin optical density estimation for HIT and LIT regions in the erythropoietic cells. A sharp light absorbance was observed at 550 nm and may be attributed to the so-called Romanowsky-DNA complex. An increase in the optical density for highly condensed chromatin at 620-680 nm (Figure 1L) correlated with differentiation, as seen for late normoblasts. Therefore, the results show some specific spectral features of chromatin that may be related to differentiation on a subcellular level at a spectral region of 550-720 nm.
Absorbance Images
On the basis of the calculated optical density of the 4 x 104 pixels of each cell, it was possible to reconstruct images defined as "absorbance images" (Figure 2A-D). Chromatin condensation in various erythroid progenitor cells was best expressed by the intensity of light absorption of the nucleus. Thus, the higher the absorbance sites in the nucleus, the more the chromatin was condensed and the cell differentiated.
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Similarity Mapping Images
Figure 2E-L reveal nuclear arrays of chromatin in a proerythroblast and in basophilic, polychromatic, and orthochromatic cells, respectively. By using a reference HIT spectra for similarity mapping, it was revealed that the major nuclear area of the proerythroblast is composed of HIT regions arranged in small unconnected patches. HIT regions in the nuclei of basophilic and polychromatic cells (Figure 2E-H) showed a circular symmetry. A central spot was observed surrounded by five or six wing-like regions distributed in a symmetrical fashion. The peripheral chromatin spots were connected to the nuclear envelope margins. The opposite complementary distribution of the LIT regions is seen in Figure 2I-L. The chromatin in the orthochromatic normoblast showed circular patches that may still indicate some ordered distribution. The same distribution has been found to exist in a large number of orthochromatic cells, as shown in Figure 3. Therefore, these spectral map images showed marked differences in chromatin condensation and distribution in the analyzed cells. Table 1 shows the values of chromatin distribution between LIT and HIT areas in differentiating erythroblasts. The LIT values depict a trend of chromatin condensation that starts at 50% of total nuclear area in erythroblasts and reaches 75% in orthochromatophilic cells.
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To determine the nature of the nuclear fragments observed in the erythroid progenitor cells in a case of AMM, we employed scanning electron microscopy (Figure 4A). The nuclear fragments appear as three separate spheres protruding from the cell surface. Transmission electron microscopy of an erythroid progenitor cell (Figure 4B) showed that the nuclear fragments were composed of a circular ring of highly condensed chromatin with an unstained center. The nuclear sorting of PI-stained progenitor red cells from the AMM patient by FACS displayed two populations of cells, one with an intact nucleus in G1-, S-, and G2-phases, and another population with fragmented DNA to the left of the G1-phase cells (Figure 4C). The cell population with fragmented DNA was collected and stained with MGG. The absorbance images of these cells, achieved by spectrally resolved image analysis as shown in Figure 5A-D, reveal a process of chromatin condensation evolving into nuclear fragmentation. Similarity mapping and image reconstruction were carried out by employing two families of spectra, one with a high absorbance peak and the other with a lower absorbance peak at 540 nm. An image of an intact nucleus reconstructed from the low absorbance peak revealed a circular distribution, as shown in Figure 5E. The similarity map of the low-absorbance complex is seen in Figure 5I, in which a circular pattern is depicted. Furthermore, fragmented nuclei showed a symmetrical, circular windmill-like pattern, as shown in Figure 5F and Figure 5J. One can note a spoke-wheel-like pattern at the nuclear periphery, shown as bright spots aimed toward a central region (Figure 5F). The nuclear fragment separated from the main nuclear body encompassed only the peripheral spots. Figure 5J shows the complementary similarity map of the low-absorbance chromatin. Figure 5G and its complementary similarity map image shown in Figure 5K reveal a nuclear fracture in an apoptotic normoblast. Figure 5H and Figure 5I clearly show the final disintegration of the apoptotic normoblast nucleus.
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Principal Component Analysis
Our interpretation of cell structure is biased by our biological education. Because the similarity mapping procedure is based on prior knowledge and identification of the different regions of chromatin, a second, more objective mathematical method was used, known as principal component analysis. Principal component analysis of May-Grunwald-Giemsa-stained cells showed that information is located in the first (largest) five eigenvalues. Each eigenvector was used for the reconstruction of a new image based on that dataset. Figure 6A-F show eigen images of AMM-derived normoblast apoptotic nuclei. The eigen images appear to be identical to the similarity mapping images of the same cells. They depict circular chromatin fragments in which the center is less condensed.
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The symmetry in the nuclei of both the differentiating erythroblasts and the AMM normoblasts confirms the assumption that chromatin organization is a highly ordered phenomenon.
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Discussion |
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In 1991, Haaf and Schmid stated that the existence of highly ordered organizational patterns in the cell nucleus appears to be beyond any doubt. It has been proposed that repetitive sequences act as a structural center for the extension and condensation of chromatin (
In the present study, Fourier transform multipixel spectroscopy and spectral imaging were used to resolve the fine nuclear morphology of differentiating and apoptotic erythroid cells stained by May-Grunwald-Giemsa. The nucleus appeared to be composed of two distinct spectral regions, the first exhibiting high intensity transmitted light with a spectral range of 550-750 nm and the second producing low-intensity transmitted spectra with a peak at 680-700 nm. This spectral variance might be explained by the difference in the local components binding the dye, such as chromatin and proteins. Optical density imaging of the cells, obtained by applying the Beer-Lambert law, produced a high-resolution picture emphasizing the difference in absorbance between the nucleus and the cytoplasm. It is well known that cytoplasmic RNA content is reduced during differentiation (
The similarity mapping procedure applied in the present study assisted in distinguishing between the different nuclear regions that were found to exhibit symmetrical patterns of circular and windmill-like rearrangements accompanying differentiation. The application of an objective mathematical algorithm for spectral image analysis, known as principal component analysis, revealed the same windmill-like structural features and symmetry. Furthermore, normoblasts in the peripheral blood of an AMM patient exhibited highly ordered condensation and fragmentation of the nuclei, characteristic of apoptosis. The applied mathematical algorithms for image analysis revealed a symmetrical breakage of the nucleus during apoptosis, with each fragment being one piece of the intact windmill pattern.
The idea of territorial organization of chromosomes was first proposed at the turn of this century by
Electron microscopic techniques during the 1960s and early 1970s failed to distinguish the hypothetical chromosome territories (
On the basis of the present results, we speculate that the circular and windmill patterns revealed by spectrally resolved imaging define a three-dimensional compartmentalization of chromatin in the differentiating nucleus. The high-intensity light transmittance regions creating the windmill pattern may represent the interchromosome domain (ICD) compartment of Zirbel (
The symmetry observed in the nuclei may be maintained by electric forces. According to
The systematizing of chromatin organization in differentiating cells and in abnormal conditions can be regarded as the most reliable tool for cytology and pathological histology. An attempt to analyze the three-dimensional nuclear structure of breast carcinomas and to correlate it with patient prognosis in a retrospective study has proved to be highly significant compared to standard histological classification (
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Acknowledgments |
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Supported by a grant from Applied Spectral Imaging, Migdal HaEmek, Israel.
We gratefully thank Ms Judith Hanania for assistance in editing the manuscript and Mr Avi Haris, Mr Jacob Langsam, and Dr Orit Katzir for their skillful assistance.
Received for publication October 21, 1996; accepted February 24, 1997.
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