Table 1 - uploaded by Irina Solovei
Content may be subject to copyright.
Example of a fluorochrome set suitable for bleed-through free five color imaging using the Leica SP2 microscope
Source publication
The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumi...
Contexts in source publication
Context 1
... illustrate this straightforward approach with differential staining of centromeres of five chromosomes in nuclei of human smooth muscle cells on paraffin sections. Images were acquired with a microscope equipped with five lasers and narrow emission filters de- signed for each channel ( Table 1 , Fig. 2 A) and showed no bleed-through in any channel. Of course, the width of emis- sion filters, together with intensity of excitation light, should be adjusted to fluorochrome and intensity of staining. ...
Context 2
... centromere probes were directly labeled with fluorochromes: centromere #1 with Cy5-dUTP; #8: Texas Red-dUTP; #17: FITC-dUTP; #18: Cy3-dUTP. Excitation wave lengths for each fluo- rochrome are indicated in the left top corner of the respective channel images (see Table 1 their emission maxima are only several nanometers apart (e.g. GFP and FITC) or they have similar wavelength range and only differ in shape. ...
Similar publications
Abstract Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of fetal bovine fibroblasts, using three-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microsco...
Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have lo...
Citations
... Axial chromatic shift correction, as well as building single grey-Frontiers in Cell and Developmental Biology frontiersin.org scale stacks, RGB-stacks, montages and maximum intensity projections was performed using ImageJ plugin StackGroom (Walter et al., 2006). The plugin is available upon request. ...
Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.
... XY pixel size varied from 20 to 120 nm. Axial chromatic shift correction, as well as building single grey-scale stacks, RGB-stacks, montages and maximum intensity projections was performed using ImageJ plugin StackGroom (Walter et al., 2006). The plugin is available upon request. ...
Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene ( Tg ). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e. for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a valuable model to study the maintenance of a high transcriptional upregulation.
SUMMARY STATEMENT
The thyroglobulin gene is highly and permanently expressed in thyrocytes of all vertebrates, at any condition and round the clock, offering a unique model to study mechanisms of high upregulation maintenance and chromatin dynamics
... It is a significant technique for study chromosomal abnormality in tumor cells. The technique has reached high level of detection sensitivity, (Individual genes can be detected), and high multiplicity (i.e.in the same nucleus several probes can be applied [2]. This delicate process consists of hybridizing a DNA probe to its complementary sequence on chromosomal preparations. ...
The objective of the study: is to investigate the correlations between the HER2 neu gene status with the clinicopathological parameters of infiltrative breast carcinoma. A total of seventy four Iraqi breast cancer patients were collected from one center (Department of Public Health) paraffin blocks were collected from histopathology department central public health laboratories, Bagdad, Iraq from 2014-2015. The cases which has been taken included invasive ductal and invasive lobular carcinoma type Women age were ranged from 24-80 years old. Evaluation of Her-2/neu gene amplification status was done using FISH and CISH techniques that showed a significant correlations with clinicopathological parameters.
... Obtained confocal stacks were processed using the ImageJ software (https://imagej.nih.gov/ij/). Before analysis, stacks were corrected for chromatic shift in z direction using the StackGroom/z-shift corrector plugin and RGB image stacks, montages of single optical sections and maximum intensity projections were generated using the StackGroom/3channels plugin (Walter et al., 2006). Images in the manuscript are mostly maximum intensity projections of several optical sections of a z-stack, unless stated otherwise. ...
Despite the well established role of nuclear organization in the regulation of gene expression, little is known about the reverse: how transcription shapes the spatial organization of the genome. In particular, given the relatively small sizes of genes and the limited resolution of light microscopy, the structure and spatial arrangement of a single transcribed gene are still poorly understood. Here, we make use of several long highly expressed mammalian genes and demonstrate that they form Transcription Loops (TLs) with polymerases moving along the loops and carrying nascent RNAs that undergo co-transcriptional splicing. TLs dynamically modify their harboring loci and extend into the nuclear interior suggesting an intrinsic stiffness. Both experimental evidence and polymer modeling support the hypothesis that TL stiffness is caused by the dense decoration of transcribed genes with multiple voluminous nascent RNPs. We propose that TL formation is a universal principle of eukaryotic gene expression.
... NA oil-immersion objective and lasers for blue (405 nm), green (488 nm), orange (561 nm), red (594 nm) and far-red (633 nm) fluorescence. Multichannel image stacks were corrected for chromatic shift and processed using a dedicated ImageJ plugin 'Stack Groom' 34 . Mice. ...
Attractions between heterochromatic regions are essential for phase separation of the active and inactive genome in inverted and conventional nuclei, whereas chromatin–lamina interactions are necessary to build the conventional genomic architecture from these segregated phases.
... Washing of unbound probe DNA, application of counterstain and visualization using fluorescence microscopy cells exist in clonal patches or as isolated events and to observe aberrations on a cell-to-cell basis rather than as a population (2). ...
... Some commercial examples for routinely used organelle-specific stains that permit the labelling of life and fixed cells alike are given in Figure 8. untreated images for each channel; arrows indicate channel -bleed through. Bottom: spectrally unmixed channels and overlay of unmixed channels [35]. Except for Cy5 all used dyes are Xanthene-derivates ...
... After Coomassie blue staining during 1h, the gels were visualized on a Chemidoc XRS+ analyzer (BioRad, right panels). PL: protein Ladders (130,100,70,55,40,35,25,15 kDa The gel electrophoresis showed that the both dyes were conjugated to the respective antibodies. During electrophoresis the obtained antibody conjugates resolved into bands with a mass of approximately 150 kDa in native conditions and two bands of 50 and 25 kDa in denaturing conditions; this corresponds to the full length antibody and heavy/light chains respectively. ...
... After Coomassie blue staining during 1h, the gels were visualized on a Chemidoc XRS+ analyzer (BioRad, right panels). PL: protein Ladders (130,100,70,55,40,35,25,15 where the excited state lifetime τs is given by the reciprocal sum of the radiative (kr) and nonradiative (knr) processes: ...
The objective of this thesis was the development and evaluation of new molecular platformsfor optical fluorescence imaging applications. This work sought to develop new tools that caneasily be modified and adapted to the specific needs of the intended use. This is required asthe fluorophore will influence the final properties and should thus be incorporated beforestructural optimization of the selected agent rather than at the very end. Two main axes wereexplored; the use of BODIPYs for the development of trackable therapeutic agents that areprimarily intended for in vitro applications and the use of azaBODIPYs for the design of an invivo compatible fluorescent platform.In the first part two fluorophores on the basis of a 3,5-dichloro-BODIPY were identified aspromising platforms. These platform molecules were selectively functionalized using a gold(I)-phosphine moiety, a thiosugar and a phosphonium to explore their selective functionalizationand investigate the influence of each substitutents position on the final properties. Weshowed that a site-specific, selective functionalization with these fragile substituents ispossible and developed 12 gold(I)-bearing therapeutic agents. We evaluated thephotophysical properties of all obtained compounds which was followed by a characterizationof their biological properties (antiproliferative properties on 3 cancer cell lines, lipophilicbalance and cellular gold accumulation as well as fluorescence imaging on 3 cell lines for upto 24h). We succeeded in developing a panel of closely related trackable compounds thatdisplay mixed activity in cells and distinct cellular localization. This investigation permitted theselection of three to four hits that will be studied further.In the second part we developed an in vivo-compatible multifunctional platform following twostrategies: the first was the use of 1,7-di(phenol)-3,5-di(phenyl)-azaBODIPY and thefunctionalization of the hydroxy groups for the development of a bioconjugable NIR-I probe.Unfortunately the developed probe displayed very unfavourable optical properties; wetherefore developed a new strategy that is entirely based on the functionalization of the boronatom. Using this approach we successfully synthesized 2 watersoluble, strongly fluorescent(NIR-I) molecular platforms that were conjugated to an innovative antibody to image the PD-L1 ligand. The developed probes displayed excellent optical properties, are stable for at least48h in mice plasma and were validated in a preclinical study on mice. The developed probesdisplayed strong fluorescence in vivo and showed no acute toxicity.The developed methodology shows great potential for further investigations and futurestudies; it can be transposed onto other closely related fluorophores and permits versatilefunctionalization with a large variety of compounds of interest. Its use is thus not limited tobiological, biochemical and medical applications.
... The missing link between these 2 methods involves imaging all chromosomes at single-cell resolution to gain information about overall organization, including which chromosomes are adjacent to or secluded from one another. This technique has been accomplished using DNA-painting probes, such as SKY paint [7,8]. ...
... Nuclei were scanned with an axial distance of 200 nm between consecutive light optical sections yielding separate stacks of 8-bit gray-scale images for each fluorescence channel with a pixel size of 60 nm. For each optical section, images were collected sequentially for all fluorochromes used, followed by correction of the axial chromatic shift for each fluorescence channel as described by Walter et al. 23 Confocal image stacks were processed with ImageJ software (http://rsb.info.nih.gov/ij) using the deconvolution plugin to enhance resolution. ...
Human myelopoiesis is an intriguing biological process during which multipotent stem cells limit their differentiation potential generating precursors that evolve into terminally differentiated cells. The differentiation process is correlated with differential gene expression and changes in nuclear architecture. In interphase, chromosomes are distinct entities known as chromosome territories and they show a radial localization that could result in a constrain of inter-homologous distance. This element plays a role in genome stability and gene expression. Here, we provide the first experimental evidence of 3D chromosomal arrangement considering two steps of human normal myelopoiesis. Specifically, multicolor 3D-FISH and 3D image analysis revealed that, in both normal human hematopoietic stem cells and myelod precursors CD14-, chromosomal position is correlated with gene density. However, we observed that inter-homologue distances are totally different during differentiation. This could be associated with differential gene expression that we found comparing the two cell types. Our results disclose an unprecedented framework relevant for deciphering the genomic mechanisms at the base of normal human myelopoiesis.
... At the forefront of studying chromatin/chromosome function, research on interphase chromatin domains and their interaction is very active [29,72,73], especially when combined with technologies to monitor DNA-protein and protein-protein interactions in living cells. For example, GFP fusion proteins, FLIP (fluorescence loss in photobleaching) or FRAP (fluorescence recovery after photobleaching), FRET (fluorescence resonance energy transfer), and FCS (fluorescence correlation spectroscopy) were used for this purpose [74,75]. ...
Background: The postgenomic era is featured by massive data collection and analyses from various large scale-omics studies. Despite the promising capability of systems biology and bioinformatics to handle large data sets, data interpretation, especially the translation of -omics data into clinical implications, has been challenging.
Discussion: In this perspective, some important conceptual and technological limitations of current systems biology are discussed in the context of the ultimate importance of the genome beyond the collection of all genes. Following a brief summary of the contributions of molecular cytogenetics/cytogenomics in the pre- and post-genomic eras, new challenges for postgenomic research are discussed. Such discussion leads to a call to search for a new conceptual framework and holistic methodologies.
Conclusion: Throughout this synthesis, the genome theory of somatic cell evolution is highlighted in contrast to gene theory, which ignores the karyotype-mediated higher level of genetic information. Since “system inheritance” is defined by the genome context (gene content and genomic topology) while “parts inheritance” is defined by genes/epigenes, molecular cytogenetics and cytogenomics (which directly study genome structure, function, alteration and evolution) will play important roles in this postgenomic era.
