Spektrale Präzisionsdistanzmikroskopie in der Genomforschung

Cremer, Christoph; Edelmann, Peter; Esa, Arif; Bornfleth, Harald; Schneider, Bernhard; Bradl, Joachim; Rinke, Bernd; Trakhtenbrot, Luba; Dietzel, Steffen; Hausmann, Michael; Cremer, Thomas

doi:10.1016/s0939-3889(15)70223-0

Cited by 8 publications

(8 citation statements)

References 22 publications

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“…This report describes the technique of spectral precision distance microscopy (SPDM)(Cremer et al ., 1996a; Cremer et al ., 1999a, b) together with in situ chromatic shift calibration using three‐colour labelling strategy of DNA sequences in intact cell nuclei. SPDM is a technique that, in principle, can be adapted to all types of fluorescence far field light microscopes that are able to record quantitatively fluorescence data of different spectral signatures (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…This calibration can be done, for instance, by means of multispectral beads (provided for example by TetraSpec, Molecular Probes, Germany). Measurements with such multispectral beads revealed that lateral shifts varied over the field of view; however, the mean absolute values were found to be smaller than 15 nm in the x ‐direction and smaller than 33 nm in the y ‐direction for all combinations of fluorochromes (Cremer et al ., 1999b). Preliminary data of distance measurements in clinical specimens based on bead calibration methods (Hausmann et al ., 1999) indicated that the precision of distance measurements can be increased using in situ (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…This experimental approach has been called spectral precision distance microscopy (SPDM). Its fundamental principles mean that it can be applied to all kinds of fluorescence far field microscopes including the above‐mentioned PSF engineering approaches (Cremer et al ., 1996a; 1999a, b; Hausmann et al ., 1999). The technique has the potential to overcome conventional resolution limits in 3D distance measurements by at least one order of magnitude.…”

Section: Introductionmentioning

confidence: 99%

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Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Esa

Edelmann

Kreth

et al. 2000

Journal of Microscopy

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107

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SummaryTopological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a`resolution equivalent', many times smaller than the conventional optical resolution.Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Esa

Edelmann

Kreth

et al. 2000

Journal of Microscopy

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107

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“…SMLM is one of the recently developed methods of super-resolution microscopy derived from spectral precision distance microscopy which was invented about 25 years ago [ 71 , 72 , 73 ]. In addition to the molecular optical resolution, the huge advantage of SMLM can be seen in its user friendliness and application versatility—the method utilizes the same technique of sample preparation and labeling as the standard optical widefield/confocal microscopy [ 54 , 55 , 58 , 67 , 74 ].…”

Section: Introductionmentioning

confidence: 99%

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

Hausmann

Falk

Neitzel

et al. 2021

IJMS

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In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

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“…Diffraction and other non-ideal optical conditions of biological specimens like variations of the refraction index limit the spatial resolution of a far-field light microscope [51,53] in practice to about 250 nm or even worse [15,28]. A general approach to overcome this limitation in fluorescence far-field microscopy is spectral precision distance microscopy [10], a technique that has been successfully applied in cytogenetics (e.g., [17]). This technique, however, does not image structural contrast modulation and limits the attainable information to objects labelled by fluorochromes.…”

Section: Introductionmentioning

confidence: 99%

Variations in Cell Surfaces of Estrogen Treated Breast Cancer Cells Detected by A Combined Instrument for Far‐Field and Near‐Field Microscopy

Perner¹,

Rapp²,

Dressler

et al. 2002

Analytical Cellular Pathology

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The response of single breast cancer cells (cell line T‐47D) to 17β‐estradiol (E2) under different concentrations was studied by using an instrument that allows to combine far‐field light microscopy with high resolution scanning near‐field (AFM/SNOM) microscopy on the same cell. Different concentrations of E2 induce clearly different effects as well on cellular shape (in classical bright‐field imaging) as on surface topography (atomic force imaging) and absorbance (near‐field light transmission imaging). The differences range from a polygonal shape at zero via a roughly spherical shape at physiological up to a spindle‐like shape at un‐physiologically high concentrations. The surface topography of untreated control cells was found to be regular and smooth with small overall height modulations. At physiological E2 concentrations the surfaces became increasingly jagged as detected by an increase in membrane height. After application of the un‐physiological high E2 concentration the cell surface structures appeared to be smoother again with an irregular fine structure. The general behaviour of dose dependent differences was also found in the near‐field light transmission images. In order to quantify the treatment effects, line scans through the normalised topography images were drawn and a rate of co‐localisation between high topography and high transmission areas was calculated. The cell biological aspects of these observations are, so far, not studied in detail but measurements on single cells offer new perspectives to be empirically used in diagnosis and therapy control of breast cancers.

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Spektrale Präzisionsdistanzmikroskopie in der Genomforschung

Cited by 8 publications

References 22 publications

Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

Variations in Cell Surfaces of Estrogen Treated Breast Cancer Cells Detected by A Combined Instrument for Far‐Field and Near‐Field Microscopy

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