Hybrid Detectors Improved Time-Lapse Confocal Microscopy of PML and 53BP1 Nuclear Body Colocalization in DNA Lesions

Foltánková, Veronika; Matula, Pavel; Sorokin, Dmitry V.; Kozubek, Stanislav; Bártová, Eva

doi:10.1017/s1431927612014353

Cited by 24 publications

(32 citation statements)

References 44 publications

(82 reference statements)

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“…For the multiscale approach (32) by Sorokin, we used Matlab code provided by the author of the method. …”

Section: Evaluated Methodsmentioning

confidence: 99%

“…A method based on a multiscale approach was described by Sorokin in (32). The first step of the approach is the calculation of the eigenvalues of the Hessian matrix for every voxel of an image at different scales r i , where r i = 2r i-1 for i = 1,. .…”

Section: Original Articlementioning

confidence: 99%

“…Adaptive thresholding methods analyzing the spot height and its size were proposed in (26-28). Other methods utilize multiscale approaches, wavelets or Fourier space decomposition to identify the spots (4,(29)(30)(31)(32). Fluorescence spots can also be detected and localized based on finding local intensity maxima and subsequent model fitting (13,33,34).…”

Section: Introductionmentioning

confidence: 99%

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Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

Štěpka

Matula

et al. 2015

Cytometry Pt A

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Reliable 3D detection of diffraction-limited spots in fluorescence microscopy images is an important task in subcellular observation. Generally, fluorescence microscopy images are heavily degraded by noise and non-specifically stained background, making reliable detection a challenging task. In this work, we have studied the performance and parameter sensitivity of eight recent methods for 3D spot detection. The study is based on both 3D synthetic image data and 3D real confocal microscopy images. The synthetic images were generated using a simulator modeling the complete imaging setup, including the optical path as well as the image acquisition process. We studied the detection performance and parameter sensitivity under different noise levels and under the influence of uneven background signal. To evaluate the parameter sensitivity, we propose a novel measure based on the gradient magnitude of the F 1 score. We measured the success rate of the individual methods for different types of the image data and found that the type of image degradation is an important factor. Using the F 1 score and the newly proposed sensitivity measure, we found that the parameter sensitivity is not necessarily proportional to the success rate of a method. This also provided an explanation why the best performing method for synthetic data was outperformed by other methods when applied to the real microscopy images. On the basis of the results obtained, we conclude with the recommendation of the HDome method for data with relatively low variations in quality, or the Sorokin method for image sets in which the quality varies more. We also provide alternative recommendations for high-quality images, and for situations in which detailed parameter tuning might be deemed expensive. V C 2015 International Society for Advancement of Cytometry Key terms fluorescence microscopy; 3D imaging; diffraction-limited spot detection; parameter sensitivity RELIABLE spot detection in 3D confocal microscopy is an important task in cell observation. The objects of interest such as proteins or other biomolecules (e.g., sequences of nucleic acids) are typically fluorescently tagged and they appear as bright, diffraction-limited particles in 3D images. For example, the proteins of interest can be genetically modified to become fluorescent while keeping their function (1), or fluorescent antibodies can be attached to proteins (2). The sequences of nucleic acids can be labeled by Fluorescence In-Situ Hybridization (FISH), which allows us to visualize parts of chromosomes such as telomeres or centromeres or even individual genes (3). It is known that the spatial distribution of subcellular objects is related to their function, and thus reliable 3D spot detection is of paramount importance in many biological applications, for example, in particle tracking (4), studies on the spatial arrangement of genes (5-7), in telomere studies (8-11), kinetochore studies (12), and in co-localization studies (13). Reliable spot detection is also essential in the Photoac...

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“…For the multiscale approach (32) by Sorokin, we used Matlab code provided by the author of the method. …”

Section: Evaluated Methodsmentioning

confidence: 99%

Section: Original Articlementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

Štěpka

Matula

et al. 2015

Cytometry Pt A

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“…[28][29][30][31] NBs such as paraspeckles, promyelocytic leukemia body (PML body) and 53BP1 nuclear body were only characterized in mammalian cells. [32][33][34] Some plant-specific NBs are also characterized, such as nuclear dicing bodies (D-bodies), photobodies, cyclophilin-containing bodies, and AKIP1-concentrated bodies. [35][36][37] NBs may be reaction sites, promoting cellular processes by concentrating proteins or RNA required, such as the nucleolus and CBs.…”

Section: Nots Assay For the Assembly Of Nuclear Bodiesmentioning

confidence: 99%

Nucleolus-tethering system (NoTS)

Liu

Fang

2014

Nucleus

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“…According to Folt ankov a et al, 14 the nucleolar DNA damage response (DDR) is strikingly different in UV-irradiated and g-irradiated genomes compared to non-irradiated cells. Thus, information regarding the radiation-specific reorganization of DNA damage-related nucleolar proteins is valuable.…”

Section: Introductionmentioning

confidence: 99%

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

Sorokin

Stixová

Sehnalová

et al. 2015

Nucleus

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(2015) Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle, Nucleus, 6:4, 301-313, DOI: 10.1080/19491034.2015 To link to this article: https://doi.org/10. 1080/19491034.2015 Abbreviations: 53BP1, p53-binding protein; ACT-D, actinomycin D; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related protein; AR, area ratio (area of nucleolus/area of nucleus); BrdU, 5-bromo-2 0 -deoxy-uridine; BER, base excision repair; CBs, Cajal bodies; DDR, DNA damage response; CPDs, cyclobutane pyrimidine dimers; DMEM, Dulbecco's modified Eagle's medium; DNA-PK, DNA-dependent protein kinase; DSBs, double-strand breaks; FA, foci area; NF, number of foci; FI, fluorescence intensity; FRAP, fluorescence recovery after photobleaching; GGR, global genome repair; GFP, green fluorescence protein; HP1, heterochromatin protein 1; HR, homologous recombination; HRR, homologous recombination repair; iMEFs, immortalized mouse embryonic fibroblasts; LR, local radius; NBs, nuclear bodies; NER, nucleotide excision repair; NHEJ, non-homologous end-joining; nA, nucleolus area; NA, nucleus area; PBS, phosphate-buffered saline; P2A, compactness; PCR, polymerase chain reaction; PML, promyelocytic leukemia bodies; rDNA, ribosomal DNA; RNA Pol II, RNA polymerase II; ROI, region of interest; RPA, replicationrelated protein A; SEM, standard error of the mean; SDS, sodium dodecyl sulfate; TCR, transcription-coupled NER; UBF1, upstream binding factor 1; UV, ultraviolet.The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or g-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of g-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that g-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli rearrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

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Hybrid Detectors Improved Time-Lapse Confocal Microscopy of PML and 53BP1 Nuclear Body Colocalization in DNA Lesions

Cited by 24 publications

References 44 publications

Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

Nucleolus-tethering system (NoTS)

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

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