Live-cell microscopy of single nuclear chromosomes and genome compartments: evaluation of labeling procedure and imaging conditions.

BACKGROUND: Single chromosomes and genome compartments in nuclei of living mammalian cells can be analyzed microscopically after specific labeling with fluorescent dyes. This is achieved by incorporating fluorescent nucleotides into the chromosomal DNA during replication (Zink et al.: Hum Genet 102:241-251, 1998; Manders et al.: J Cell Biol 144:813-821, 1999; Sadoni et al.: J Cell Biol 146:1211-1226, 1999). We characterized the potential artificial impact of this approach on chromosome structure and dynamics. We also evaluated potential sources of artifacts in corresponding live-cell imaging. MATERIALS AND METHODS: The subchromosomal distribution of labeled DNA was analyzed, and the fate of labeled nucleotides within cell nuclei was studied. Cell-cycle parameters were used to analyze cell function after incorporation of fluorescent nucleotides. The influences of phototoxic effects on cell division and morphology were studied. RESULTS: Fluorescent nucleotides were only incorporated for a restricted time period during S-phase, and a uniform labeling of chromosomal DNA could not be achieved. Fluorescent nucleotides incorporated into the DNA showed no or only mild effects on cell growth. Cell-cycle parameters and cellular morphology were valuable indicators for proper cell function during live-cell imaging. CONCLUSIONS: There is no indication for a substantial impairment of cellular functions if fluorochromes are covalently linked to chromosomal DNA. The controls we present for proper cell function during the imaging period are of general importance, as appropriate controls for live cell microscopy have not yet been well-defined.     

Multiple topological labeling for imaging single plasmids

Sequence-specific labeling methods for double-stranded DNA are required for mapping protein binding sites or specific DNA structures on circular DNA molecules by high-resolution imaging techniques such as electron and atomic force microscopies. Site-specific labeling can be achieved by ligating a DNA fragment to a stem-loop-triplex-forming oligonucleotide, thereby forming a topologically linked complex. The superhelicity of the plasmid is not altered and the process can be applied to two different target sites simultaneously, using DNA fragments of different sizes. Observation of the labeled plasmids by electron microscopy revealed that, under conditions where the triple helices were stable, the two labels were located at 339+/-34 bp from one another, in agreement with the distance between the two target sequences for triple helix formation (350 bp). Under conditions where the triple helices were not stable, the short DNA fragments could slide away from their target site. The concomitant attachment of two different stable labels makes it possible, for the first time to our knowledge, to label a circular DNA molecule and obtain information on its direction. In addition to its potential applications as a tool for structural investigations of single DNA molecules and their interactions with proteins, this DNA labeling method may also prove useful in biotechnology and gene therapy.     

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.     

Identifying conformational changes with site-directed spin labeling

Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins. This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure. The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of alpha-helices and beta-strands (ii) relative movements of domains and (iii) changes in secondary structure. Such events can be monitored in the millisecond time-scale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins that can be investigated, and only 50-100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems.     

Flow cytometric evaluation of sperm parameters in relation to fertility potential

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.     

Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy

An atypical peg-like terminal constriction (“peg”) on metaphase chromosomes of the plant genus Oziroë could be identified as a nucleolus organizing region (NOR) by detecting 45S rDNA with correlative light microscopy (LM) and scanning electron microscopy (SEM) in situ hybridization (ISH). Using high-resolution 3D analytical SEM, the architecture and DNA distribution of the peg-like NOR were characterized as typical for chromosomes, albeit with significantly smaller chromomeres. ISH procedure was improved for SEM concerning signal localization, labeling efficiency, and structural preservation, allowing 3D SEM analysis of the peg-like NOR structure and rDNA distribution for the first time. It could be shown that implementation of FluoroNanogold markers is an attractive tool that allows efficient immunodection in both LM and SEM. A model is proposed for the peg structure and its mode of condensation.     

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.     

Chromosomal in situ hybridization using yeast artificial chromosomes

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.     

A fast,low cost,and highly efficient fluorescent DNA labeling method using methyl green

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.     

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

To genuinely understand how complex biological structures function, we must integrate knowledge of their dynamic behavior and of their molecular machinery. The combined use of light or laser microscopy and electron microscopy has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, is often referred to as ‘correlative microscopy’. Correlative imaging allows researchers to gain additional novel structure–function information, and such information provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or ‘combined’) microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches. In this topical review, we provide a brief historical perspective of correlative microscopy and an in-depth overview of correlative sample-preparation and imaging methods presently available, including future perspectives on the trend towards integrative microscopy and microanalysis.     

Fluorescence Tracing of Intracellular Proteins

Developments in fluorescence microscopy and the availability of fluorescently labeled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci allowing deep insight into cell and organelle biology. Furthermore, confocal microscopy permits analysis of the three dimensional architecture of cells that could not be accomplished by conventional light microscopy. The goal of fluorescence protein tracing by microscopy is to visualize cellular constituents and general cytoarchitecture as close to native organization as possible. To achieve this, and to preserve cellular structure in the best possible manner, the specimen is usually fixed chemically. Here I review several standard fixation, permeabilization and labeling schemes followed by examples of several standard imaging techniques.     

H-1 parvovirus-associated replication bodies: a distinct virus-induced nuclear structure

We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells.     

Tyramide Signal Amplification Method in Multiple-Label Immunofluorescence Confocal Microscopy

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.     

Method of specific detection of apoptosis using formamide-induced DNA denaturation assay.

We compared the reliability between apoptosis detection methods, namely, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method and formamide-induced DNA denaturation assay using a monoclonal antibody (MAb) to single-stranded DNA (ssDNA) (formamide-MAb assay). Reaction targets in these methods are different: the TUNEL method recognizes free 3'-OH DNA ends, whereas the formamide-MAb assay detects ssDNA itself (25-30 bp). We found that the formamide-MAb assay immunohistochemically detected apoptotic cells, whereas the TUNEL method detected apoptotic cells as well as mitotic and necrotic cells. The TUNEL method recognized not only 3'-OH DNA ends cleaved by DNase during apoptosis but also constitutive physiological nicking that occurs in DNA duplication and histone posttranslational modifications during mitosis and random DNA breaks during necrotic execution. By electron microscopy, the mean labeling density (the number of 3'-OH DNA ends/nuclear area) obtained by the TUNEL method was determined to be consistently higher than that (the number of ssDNAs/nuclear area) obtained by the formamide-MAb assay. On the basis of these findings, we conclude that the formamide-MAb assay was more specific than the TUNEL method for the detection of apoptotic cells using electron microscopy; however, the labeling intensity of the formamide-MAb assay was slightly weaker than that of the TUNEL method.     

The RecB subunit of the Escherichia coli RecBCD enzyme couples ATP hydrolysis to DNA unwinding.

The RecB subunit of the Escherichia coli RecBCD enzyme has previously been reported to possess DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). Here we demonstrate that a specific interaction between RecB protein and ATP can also be shown by photoaffinity labeling with the ATP analogue 8-azido-ATP. Furthermore, the capacity of the RecB protein to support ATP hydrolysis varies with the structure and length of the DNA cofactor. Single-stranded linear and circular DNA are markedly better in promoting ATP hydrolysis than duplex DNA. The purified RecB protein can function as a DNA helicase, displacing oligonucleotides annealed to viral M13 DNA in an ATP-dependent and orientation-specific manner.     

5-溴脱氧尿嘧啶核苷标记酵母基因组DNA的方法

胸腺嘧啶类似物5-溴脱氧尿嘧啶核苷(BrdU)标记技术是一种研究DNA复制、修复等生命过程的有效手段。由于酿酒酵母(Saccharomyces cerevisiae)中缺少胸腺嘧啶核苷酸补救途径,胞外BrdU不能有效的渗入到基因组中,使该技术在酿酒酵母中的应用受到极大制约。通过在基因组中引入单纯疱疹病毒胞苷激酶(HSV-TK)和人类平衡核苷转运蛋白(hENT1)基因,工作建立了BrdU标记酵母基因组DNA的方法。在生长对数中期加入0.2mg/ml BrdU,离体检测法检测发现,标记3h的荧光信号较1h、5h时强;胞内检测法结果显示,标记3h时55.3%的基因组DNA中能够渗入BrdU。该工作为酿酒酵母DNA复制、修复等方面提供了直接有效的研究方法。     

Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells(1). DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique(2-4). In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.     

The use of FITC-conjugated monoclonal antibodies for determination of S-phase cells with fluorescence microscopy

A method is modified to determine the DNA synthesizing cells in primary human breast tumors and cells with idiopathic thrombocytopenic purpura (ITP) with FITC-conjugated monoclonal antibody against bromodeoxyuridine (FITC-M-anti-BrdUrd) and fluorescence microscopy. The DNA synthesizing cells were also determined from a portion of the same tissues by classical tritiated thymidine labeling (3HdThd) and autoradiography. The results from bromodeoxyuridine labeling index (BrdUrd-LI) and tritiated thymidine labeling index (3HdThd-LI) obtained from the same tissues were compared. The mean BrdUrd-LI for breast tumor was 5.4 +/- 1.0% and the mean 3HdThd-LI was 5.5 +/- 1.1%. Similarly, the labeling indexes obtained from mononuclear leukocytes of healthy donors had means of 0.5 +/- 0.1% and 0.6 +/- 0.1% for BrdUrd-LI and 3HdThd-LI, respectively. The change in the proliferation rate of mononuclear leukocyte population in the samples obtained from patients with ITP could be observed by both methods. The mean BrdUrd-LI of mononuclear leukocytes for this hematological disorder was 5.4 +/- 0.8%. These results suggest that was 6.1 +/- 0.8%. These results suggest that this relatively simple technique offers an alternative method for determining the DNA synthesizing cells in a given cell population.     

Nonlinear microscopy: new techniques and applications

Nonlinear microscopy, a general term that embraces any microscopy technique based on nonlinear optics, is further establishing itself as an important tool in neurobiology. Recent advances in labels, labeling techniques, and the use of native or genetically encoded contrast agents have bolstered the capacity of nonlinear microscopes to image the structure and function of not just single cells but of entire networks of cells. Along with novel strategies to image over exceptionally long durations and with increased depth penetration in living brains, these advances are opening new opportunities in neurobiology that were previously unavailable.