Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

Risk of Ischemic Stroke and Transient Ischemic Attack Is Increased up to 90 Days after Non-Carotid and Non-Cardiac Surgery.

BACKGROUND: The risk of stroke after cardiac and carotid surgery is well established. In contrast, stroke risk in association with non-cardiac and non-carotid surgery and its time course are insufficiently known. We investigated the prevalence of recent and planned surgery among patients with stroke and transient ischemic attack (TIA), time dependency of stroke risk, stroke etiology, and interruption of antithrombotic medication in association with surgery. METHODS: Data on type and date of surgery and similar interventions within the last year or planned for the next 2 weeks were anonymously collected together with demographic data, vascular risk factors, stroke severity, handicap before stroke and stroke etiology within a state-wide, mandatory, hospital-based acute stroke care quality monitoring project (Rhineland-Palatinate, Germany) for 1 year (2010). RESULTS: Non-carotid and non-cardiothoracic surgery was reported as performed within 1 year before the index event or as planned for the next 2 weeks thereafter in 532 out of 12,120 patients with ischemic stroke/TIA (4.4%). Compared to 91-365 days before stroke/TIA as reference period, risk of cerebral ischemia (per day analysis) was increased for surgery within 61-90 days before ischemia (rate ratio 2.0, 95% CI 1.5-2.8) and continuously increased along shorter intervals between stroke and surgery (31-60 days: rate ratio 3.6, 95% CI 2.9-4.5; 15-30 days: rate ratio 8.2, 95% CI 6.7-10.1; 8-14 days: rate ratio 13.2, 95% CI 10.3-16.8; 4-7 days: rate ratio 16.5, 95% CI 12.2-22.1) peaking at an interval of 1-3 days before ischemia (rate ratio 34.0, 95% CI 26.9-42.8). On the day of surgery, rate ratio was 14.8 (95% CI 7.8-27.9) and for planned surgery it was 2.7 (95% CI 1.8-4.0). Results were similar for first-ever and for recurrent ischemic stroke. Perioperative stroke/TIA was positively associated with atrial fibrillation and cardioembolic stroke etiology, higher mortality, more severe neurological deficits at discharge, and longer hospital stay; and it was inversely associated with microangiopathic etiology and discharge at home. In 34.5% of patients with recent/planned surgery, prior antithrombotic or anticoagulant medication had been interrupted. CONCLUSIONS: Recent or planned surgery imposes a considerable short-term stroke risk particularly by cardioembolism with cessation of medication as an important contributor. Stroke after surgery is associated with poor outcome and high mortality. Better strategies to reduce the burden of perioperative stroke are urgently required.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy.

Volume increase and spatial shifts of chromosome territories in nuclei of radiation-induced polyploidizing tumour cells.

The exposure of tumour cells to high doses of ionizing radiation can induce endopolyploidization as an escape route from cell death. This strategy generally results in mitotic catastrophe during the first few days after irradiation. However, some cells escape mitotic catastrophe, polyploidize and attempt to undergo genome reduction and de-polyploidization in order to create new, viable para-diploid tumour cell sub-clones. In search for the consequences of ionizing radiation induced endopolyploidization, genome and chromosome architecture in nuclei of polyploid tumour cells, and sub-nuclei after division of bi- or multi-nucleated cells were investigated during 7 days following irradiation. Polyploidization was induced in p53-function deficient HeLa cells by exposure to 10Gy of X-irradiation. Chromosome territories #1, #4, #12 and centromeres of chromosomes #6, #10, #X were labelled by FISH and analysed for chromosome numbers, volumes and spatial distribution during 7 days post irradiation. The numbers of interphase chromosome territories or centromeres, respectively, the positions of the most peripherally and centrally located chromosome territories, and the territory volumes were compared to non-irradiated controls over this time course. Nuclei with three copies of several chromosomes (#1, #6, #10, #12, #X) were found in the irradiated as well as non-irradiated specimens. From day 2 to day 5 post irradiation, chromosome territories (#1, #4, #12) shifted towards the nuclear periphery and their volumes increased 16- to 25-fold. Consequently, chromosome territories returned towards the nuclear centre during day 6 and 7 post irradiation. In comparison to non-irradiated cells (∼500µm(3)), the nuclear volume of irradiated cells was increased 8-fold (to ∼4000µm(3)) at day 7 post irradiation. Additionally, smaller cell nuclei with an average volume of about ∼255µm(3) were detected on day 7. The data suggest a radiation-induced generation of large intra-nuclear chromosome territories and their repositioning prior to genome reduction.

PML promotes MHC class II gene expression by stabilizing the class II transactivator.

Promyelocytic leukemia (PML) nuclear bodies selectively associate with transcriptionally active genomic regions, including the gene-rich major histocompatibility (MHC) locus. In this paper, we have explored potential links between PML and interferon (IFN)-γ-induced MHC class II expression. IFN-γ induced a substantial increase in the spatial proximity between PML bodies and the MHC class II gene cluster in different human cell types. Knockdown experiments show that PML is required for efficient IFN-γ-induced MHC II gene transcription through regulation of the class II transactivator (CIITA). PML mediates this function through protection of CIITA from proteasomal degradation. We also show that PML isoform II specifically forms a stable complex with CIITA at PML bodies. These observations establish PML as a coregulator of IFN-γ-induced MHC class II expression.

Nuclear position and shape deformation of chromosome 8 territories in pancreatic ductal adenocarcinoma.

Cell type specific radial positioning of chromosome territories (CTs) and their sub-domains in the interphase seem to have functional relevance in non-neoplastic human nuclei, while much less is known about nuclear architecture in carcinoma cells and its development during tumor progression. We analyzed the 3D-architecture of the chromosome 8 territory (CT8) in carcinoma and corresponding non-neoplastic ductal pancreatic epithelium. Fluorescence-in-situ-hybridization (FISH) with whole chromosome painting (WCP) probes on sections from formalin-fixed, paraffin wax-embedded tissues from six patients with ductal adenocarcinoma of the pancreas was used. Radial positions and shape parameters of CT8 were analyzed by 3D-microscopy. None of the parameters showed significant inter-individual changes. CT8 was localized in the nuclear periphery in carcinoma cells and normal ductal epithelial cells. Normalized volume and surface of CT8 did not differ significantly. In contrast, the normalized roundness was significantly lower in carcinoma cells, implying an elongation of neoplastic cell nuclei. Unexpectedly, radial positioning of CT8, a dominant parameter of nuclear architecture, did not change significantly when comparing neoplastic with non-neoplastic cells. A significant deformation of CT8, however, accompanies nuclear atypia of carcinoma cells. This decreased roundness of CTs may reflect the genomic and transcriptional alterations in carcinoma.

COMBO-FISH enables high precision localization microscopy as a prerequisite for nanostructure analysis of genome loci.

With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.

COMBinatorial Oligo FISH: directed labeling of specific genome domains in differentially fixed cell material and live cells.

With the improvement and completeness of genome databases, it has become possible to develop a novel fluorescence in situ hybridization (FISH) technique called COMBinatorial Oligo FISH (COMBO-FISH). In contrast to other (standard) FISH applications, COMBO-FISH makes use of a bioinformatic approach for probe set design. By means of computer genome database search, oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that they all colocalize within a given genome (gene) target. Typically, probe sets of about 20-40 stretches are designed within 50-250 kb, which is enough to get an increased fluorescence signal specifically highlighting the target from the background. Although "specific colocalization" is the only necessary condition for probe selection, i.e. the probes of different lengths can be composed of purines and pyrimidines, we additionally refined the design strategy restricting the probe sets to homopurine or homopyrimidine oligonucleotides so that depending on the probe orientation either double (requiring denaturation of the target double strand) or triple (omitting denaturation of the target strand) strand bonding of the probes is possible. The probes used for the protocols described below are DNA or PNA oligonucleotides, which can be synthesized by established automatized techniques. We describe different protocols that were successfully applied to label gene targets via double- or triple-strand bonding in fixed lymphocyte cell cultures, bone marrow smears, and formalin-fixed, paraffin-wax embedded tissue sections. In addition, we present a procedure of probe microinjection in living cells resulting in specific labeling when microscopically detected after fixation.

Visualization, analysis, and design of COMBO-FISH probes in the grid-based GLOBE 3D genome platform.

The genome architecture in cell nuclei plays an important role in modern microscopy for the monitoring of medical diagnosis and therapy since changes of function and dynamics of genes are interlinked with changing geometrical parameters. The planning of corresponding diagnostic experiments and their imaging is a complex and often interactive IT intensive challenge and thus makes high-performance grids a necessity. To detect genetic changes we recently developed a new form of fluorescence in situ hybridization (FISH) - COMBinatorial Oligonucleotide FISH (COMBO-FISH) - which labels small nucleotide sequences clustering at a desired genomic location. To achieve a unique hybridization spot other side clusters have to be excluded. Therefore, we have designed an interactive pipeline using the grid-based GLOBE 3D Genome Viewer and Platform to design and display different labelling variants of candidate probe sets. Thus, we have created a grid-based virtual "paper" tool for easy interactive calculation, analysis, management, and representation for COMBO-FISH probe design with many an advantage: Since all the calculations and analysis run in a grid, one can instantly and with great visual ease locate duplications of gene subsequences to guide the elimination of side clustering sequences during the probe design process, as well as get at least an impression of the 3D architectural embedding of the respective chromosome region, which is of major importance to estimate the hybridization probe dynamics. Beyond, even several people at different locations could work on the same process in a team wise manner. Consequently, we present how a complex interactive process can profit from grid infrastructure technology using our unique GLOBE 3D Genome Platform gateway towards a real interactive curative diagnosis planning and therapy monitoring.

Cell nucleus architecture in health and medicine: geometrical descriptors and their use in grid based case studies.

Adopting the world wide accessible Grid computing power and data management structures enables usage of large image data bases for individual diagnosis and therapy decisions. Here, we define several descriptors of the genome architecture of cell nuclei which are the basis of a detailed analysis for conclusions on the health state of an individual patient. All these descriptors can be accessed by automatic inspection of microscopic images of fluorescently labelled nuclei, obtained from cells from tissue sections or blood and subjected to standard biochemical protocols. We demonstrate how the combinatorial, geometrical and statistical parameters may be used in diagnosis and therapy monitoring.

Quality monitoring of acute stroke care in Rhineland-Palatinate, Germany, 2001-2006.

BACKGROUND AND PURPOSE: Quality monitoring projects are useful tools to improve the quality and to assess temporal trends of stroke care in larger populations. METHODS: In Rhineland-Palatinate, Germany, a statewide, hospital-based, acute stroke care quality monitoring project was started in 2001. Initially, participation was mandatory for all hospitals with dedicated stroke units and from 2006 onward was mandatory for all hospitals. Quality monitoring included a structured data assessment and quality indicators for procedural measures. RESULTS: Between 2001 and 2006, the numbers of patients registered annually (N=6389 vs N=10 610), admission <3 hours after stroke onset (28.2% vs 34.6%), admission via emergency medical systems (38.1% vs 50.3%), and treatment in stroke units (44.3% vs 59.5%) increased significantly (P<0.0001, respectively). In ischemic stroke, use of thrombolytic therapy increased (for patients admitted <3 hours after onset, 6.5% vs 14.1%), whereas therapy with high-dose heparin declined (24.5% vs 6.0%, P<0.0001). Several quality indicators (performance of neuroimaging and Doppler/duplex sonography, neuroimaging <3 hours after admission) showed stable results at a high level; more patients received echocardiography (62.2% vs 74.0%), but fewer patients were rapidly examined by extracranial Doppler/duplex sonography (68.7% vs 62.8%, P<0.0001). Diagnosis and treatment of hypertension and hyperlipidemia, use of aspirin and combined aspirin/dipyridamole, and diagnosis of atrial fibrillation increased (P<0.0001, respectively). Use of oral anticoagulation remained stable at approximately 38% of patients with cardioembolism. CONCLUSIONS: Although these results reflect high standards of acute stroke care and improvements regarding early admission, thrombolytic therapy, and several secondary preventive measures, there is still the potential for further improvement regarding thrombolysis, use of oral anticoagulation and statins, and admission to stroke units, for example.

Conception of an image data base for cell nuclei and geometric algorithms for diagnosis and therapy monitoring.

Parameters of the genome architecture of cell nuclei like copy number changes of genes or numerical and structural aberrations of chromosomes displayed by changes of size, shape, form and geometric arrangement of the related territories and domains play an important role in tumour diagnosis and monitoring of tumour therapy. We have defined data structures for such parameters, accompanied by meta data describing cell biology and microscopy protocols, and developed algorithms to deduce geometric data from microscopic raw images of fluorescently labelled cell nuclei. The statistical evaluation of nucleus geometry and architecture data is a valuable aid for diagnostic decisions and monitoring of cancer development, as indicated by several research case studies. The algorithms and data storage devices are presently administrated by different operating systems. Unification of workflow is being achieved for a local cluster, but gridification is still subject to problems of licensing, monitoring, and administering systems, including data security.

Spatial allelic imbalance of BCL2 genes and chromosome 18 territories in nonneoplastic and neoplastic cervical squamous epithelium.

Several studies suggest a correlation between genome architecture and gene function. To elucidate mechanisms of gene positioning during cell differentiation and malignant transformation we investigated the nuclear positions of the BCL2 alleles and chromosome 18 territories in different layers of nonneoplastic cervical squamous epithelium and cervical squamous carcinomas in relation to gene expression. Fluorescence in situ hybridization and three-dimensional (3D) image analysis using tissue sections revealed that one BCL2 allele was located more peripherally than the other one in nuclei of the basal layer of nonneoplastic epithelium. During terminal cell differentiation the outer BCL2 allele showed a shift towards the nuclear center. In BCL2-expressing carcinomas the inner BCL2 allele was located more peripherally compared with the basal layer of nonneoplastic epithelium. Our results suggest a functional relevance of unequal allelic BCL2 gene positioning and support the hypothesis that transcriptional BCL2 activation is associated with BCL2 relocation towards the nuclear periphery.

In silico study of kinetochore control, amplification, and inhibition effects in MCC assembly.

Eukaryotic cells rely on a surveillance mechanism, the "Spindle Assembly Checkpoint"SACM in order to ensure accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. In different organisms, a mitotic checkpoint complex (MCC) composed of Mad2, Bub3, BubR1/Mad3, and Cdc20 inhibits the anaphase promoting complex (APC/C) to initiate promotion into anaphase. The mechanism of MCC formation and its regulation by the kinetochore are unclear. Here, we constructed dynamical models of MCC formation involving different kinetochore control mechanisms including amplification as well as inhibition effects, and analysed their quantitative properties. In particular, in this system, fast and stable metaphase to anaphase transition can only be triggered when the kinetochore controls the Bub3:BubR1-related reactions; signal amplification and inhibition play a subordinate role. Furthermore, when introducing experimentally determined parameter values into the models analysed here, we found that effective MCC formation is not combined with complete Cdc20 sequestering. Instead, the MCC might bind and completely block the APC/C. The SACM might function by an MCC:APC/C complex rearrangement.

Mad2 binding is not sufficient for complete Cdc20 sequestering in mitotic transition control (an in silico study).

For successful mitosis, metaphase has to be arrested until all centromeres are properly attached. The onset of anaphase, which is initiated by activating the APC, is controlled by the spindle assembly checkpoint (M)SAC. Mad2, which is a constitutive member of the (M)SAC, is supposed to inhibit the activity of the APC by sequestering away its co-activator Cdc20. Mad1 recruits Mad2 to unattached kinetochores and is compulsory for the establishment of the Mad2 and Cdc20 complexes. Recently, based on results from in vivo and in vitro studies, two biochemical models were proposed: the Template and the Exchange model. Here, we derive a mathematical description to compare the dynamical behaviour of the two models. Our simulation analysis supports the Template model. Using experimentally determined values for the model parameters, the Cdc20 concentration is reduced down to only about half. Thus, although the Template model displays good metaphase-to-anaphase switching behaviour, it is not able to completely describe (M)SAC regulation. This situation is neither improved by amplification nor by p31(comet) inhibition. We speculate that either additional reaction partners are required for total inhibition of Cdc20 or an extended mechanism has to be introduced for (M)SAC regulation.

In-silico modeling of the mitotic spindle assembly checkpoint.

BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (M)SAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (M)SAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (M)SAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (M)SAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.

Anemia as a risk factor for cerebral venous thrombosis? An old hypothesis revisited. Results of a prospective study.

BACKGROUND: Several case reports have linked iron deficiency anemia with the occurrence of cerebral venous thrombosis (CVT) or stroke, yet, it is unclear whether this is a chance association. METHODS: In a case-control design data of whole blood count and screening for thrombophilic coagulation abnormalities of 121 prospectively identified patients with CVT and 120 healthy controls were compared. Anemia was defined as a hemoglobin (Hb) concentration of <120 g/l in females, and <130 g/l in males, severe anemia as a Hb <90 g/l. Adjusted odds ratios (OR) were calculated based on a logistic regression model treating variables with a level of significance of p < or = 0.2 on univariate analysis as potential confounders. RESULTS: Thrombophilia (OR 1.22, 95% CI 1.07-1.76, p < 0.01), severe anemia (OR 1.10, 95% CI 1.01-2.22, p < 0.05), and hypercholesterinemia (OR 1.21, 95% CI 1.04-2.57, p < 0.05) were the only independent variables associated with CVT on multivariate analysis. CONCLUSION: Severe anemia is significantly and independently associated with CVT.

Spinocerebellar ataxia 14: novel mutation in exon 2 of PRKCG in a German family.

We describe a novel mutation in the gene coding for protein kinase C gamma (PRKCG) in patients of a German family affected with slowly progressive gait ataxia, dysarthria, and nystagmus. The G/T missense mutation occurred in exon 2 of PRKCG and results in a substitution of glycine by valine (G63V) in the evolutionarily highly conserved cysteine-rich region 1/C1 domain of PRKCG. Among the 20 mutations described to date, this is the first mutation located in exon 2 of PRKCG.

Comparison of triple helical COMBO-FISH and standard FISH by means of quantitative microscopic image analysis of abl/bcr positions in cell nuclei.

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an approach.