scholarly journals Measuring chromosome conformation by fluorescence microscopy

2019 ◽  
Author(s):  
Brian C. Ross ◽  
Fabio Anaclerio ◽  
Nicola Lorusso ◽  
Mario Ventura ◽  
Jim Costello
Keyword(s):  
Fluorescence Microscopy ◽  
Structural Biology ◽  
Single Cells ◽  
Reconstruction Algorithms ◽  
Computational Tools ◽  
Chromosome Conformation ◽  
Reconstruction Quality ◽  
Sequential Fish ◽  
Connecting The Dots

AbstractMeasurement of in-vivo chromosome conformations (structures) in single cells is a major technological goal of structural biology. If one could identify many genetic loci in a microscope image despite the limited palette of fluorescent colors used to label them, then the conformation could be solved at some resolution by ‘connecting the dots’. Computational tools for making this reconstruction are expected to produce near-perfect reconstructions when the number of fluorescent colors is high enough, irrespective of the number of loci assayed. Here we report the first experimental test of the performance of these reconstruction algorithms and check their ability to reconstruct experimentally-measured conformations. We also demonstrate the experimental metrics needed to assess reconstruction quality. Our results indicate that current sequential FISH experiments may be close to the point where the reconstructions are nearly flawless at some distance scales.

4D in in vivo 2-photon laser scanning fluorescence microscopy with sample motion in 6 degrees of freedom

2011 ◽  
Vol 200 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Sabine Scheibe ◽  
Mario M. Dorostkar ◽  
Christian Seebacher ◽  
Rainer Uhl ◽  
Frank Lison ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Laser Scanning ◽  
Degrees Of Freedom ◽  
Sample Motion ◽  
6 Degrees Of Freedom

In vivo expansion of cells expressing acquired CSF3R mutations in patients with severe congenital neutropenia

Blood ◽  
2009 ◽  
Vol 113 (3) ◽  
pp. 668-670 ◽  
Author(s):  
Manuela Germeshausen ◽  
Karl Welte ◽  
Matthias Ballmaier
Keyword(s):  
Single Cells ◽  
Bone Marrow Failure ◽  
Point Mutations ◽  
Predictive Marker ◽  
Lymphoid Cells ◽  
Lower Percentage ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Bone Marrow Failure Syndrome

Abstract Severe congenital neutropenia (CN) is a rare bone marrow failure syndrome with a high incidence of acute leukemia. In previous studies, we could show that point mutations in the gene for the granulocyte colony-stimulating factor (G-CSF) receptor CSF3R are a highly predictive marker for leukemic development in CN patients. To find out at which stage of hematopoietic development these mutations emerge and how they are propagated during hematopoietic differentiation, we analyzed single cells of different hematopoietic subpopulations from CN patients with CSF3R mutations. We found that CSF3R mutations are not restricted to the myeloid compartment but are also detectable in lymphoid cells, although at a much lower percentage. From our observations, we conclude that CSF3R mutations are acquired in multipotent hematopoietic progenitor cells in CN patients and that they are clonally expanded in myeloid cells expressing the G-CSF receptor due to the growth advantage mediated by the CSF3R mutation.

An In Vivo Fluorescent Sensor Reveals Intracellular Ins(1,3,4,5)P4Dynamics in Single Cells

2010 ◽  
Vol 49 (12) ◽  
pp. 2150-2153 ◽  
Author(s):  
Reiko Sakaguchi ◽  
Kazuki Tainaka ◽  
Naoko Shimada ◽  
Shun Nakano ◽  
Masafumi Inoue ◽  
...  
Keyword(s):  
Fluorescent Sensor ◽  
Single Cells

scholarly journals Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

2014 ◽  
Vol 25 (22) ◽  
pp. 3699-3708 ◽  
Author(s):  
Anyimilehidi Mazo-Vargas ◽  
Heungwon Park ◽  
Mert Aydin ◽  
Nicolas E. Buchler
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Photon Flux ◽  
Luminescence Microscopy ◽  
Cell Cycle Genes ◽  
Light Excitation ◽  
Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

scholarly journals Connecting the dots between computational tools to analyse soil–root water relations

2018 ◽  
Vol 70 (9) ◽  
pp. 2345-2357 ◽  
Author(s):  
Sixtine Passot ◽  
Valentin Couvreur ◽  
Félicien Meunier ◽  
Xavier Draye ◽  
Mathieu Javaux ◽  
...  
Keyword(s):  
Water Relations ◽  
Computational Tools ◽  
Connecting The Dots

Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa

1989 ◽  
Vol 256 (1) ◽  
pp. G254-G263 ◽  
Author(s):  
C. S. Chew ◽  
M. Ljungstrom ◽  
A. Smolka ◽  
M. R. Brown
Keyword(s):  
Gastric Mucosa ◽  
Primary Culture ◽  
Cultured Cells ◽  
Single Cells ◽  
Parietal Cells ◽  
Ultrastructural Changes ◽  
Long Term Effects ◽  
H2 Receptor Antagonist ◽  
Morphological Transformations

A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, [14C]aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor "physiological" responses at the same time within the same cell.

Structural biology of tissue factor, the initiator of thrombogenesis in vivo 1

1994 ◽  
Vol 8 (6) ◽  
pp. 385-390 ◽  
Author(s):  
Wolfram Ruf ◽  
Thomas S. Edgington
Keyword(s):  
Tissue Factor ◽  
Structural Biology
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