Quantitative Fluorescence Imaging Approach for the Study of Polyploidization in Hepatocytes

We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms.

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In Situ Bioorthogonal Metabolic Labeling for Fluorescence Imaging of Virus Infection In Vivo

Small ◽

10.1002/smll.201604036 ◽

2017 ◽

Vol 13 (17) ◽

pp. 1604036 ◽

Cited By ~ 11

Author(s):

Hong Pan ◽

Wen-jun Li ◽

Xiang-jie Yao ◽

Ya-yun Wu ◽

Lan-lan Liu ◽

...

Keyword(s):

Virus Infection ◽

Fluorescence Imaging ◽

Metabolic Labeling

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Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker

Journal of Neurosurgery ◽

10.3171/2011.2.jns101451 ◽

2011 ◽

Vol 115 (1) ◽

pp. 11-17 ◽

Cited By ~ 192

Author(s):

Pablo A. Valdés ◽

Frederic Leblond ◽

Anthony Kim ◽

Brent T. Harris ◽

Brian C. Wilson ◽

...

Keyword(s):

Brain Tissue ◽

Fluorescence Imaging ◽

Low Grade ◽

Normal Brain ◽

Intracranial Tumors ◽

Neoplastic Tissue ◽

Fluorescence Measurements ◽

Significant Difference ◽

Quantitative Fluorescence

Object Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative fluorescence of protoporphyrin IX (PpIX), synthesized endogenously following δ-aminolevulinic acid (ALA) administration, has been used for this purpose in high-grade glioma (HGG). The authors show that diagnostically significant but visually imperceptible concentrations of PpIX can be quantitatively measured in vivo and used to discriminate normal from neoplastic brain tissue across a range of tumor histologies. Methods The authors studied 14 patients with diagnoses of low-grade glioma (LGG), HGG, meningioma, and metastasis under an institutional review board–approved protocol for fluorescence-guided resection. The primary aim of the study was to compare the diagnostic capabilities of a highly sensitive, spectrally resolved quantitative fluorescence approach to conventional fluorescence imaging for detection of neoplastic tissue in vivo. Results A significant difference in the quantitative measurements of PpIX concentration occurred in all tumor groups compared with normal brain tissue. Receiver operating characteristic (ROC) curve analysis of PpIX concentration as a diagnostic variable for detection of neoplastic tissue yielded a classification efficiency of 87% (AUC = 0.95, specificity = 92%, sensitivity = 84%) compared with 66% (AUC = 0.73, specificity = 100%, sensitivity = 47%) for conventional fluorescence imaging (p < 0.0001). More than 81% (57 of 70) of the quantitative fluorescence measurements that were below the threshold of the surgeon's visual perception were classified correctly in an analysis of all tumors. Conclusions These findings are clinically profound because they demonstrate that ALA-induced PpIX is a targeting biomarker for a variety of intracranial tumors beyond HGGs. This study is the first to measure quantitative ALA-induced PpIX concentrations in vivo, and the results have broad implications for guidance during resection of intracranial tumors.

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Identification on Mantle Cell Lymphoma Using CD20 and CD5 Coupled Upconversion Fluorescent Nanoprobes

Journal of Nanomaterials ◽

10.1155/2018/3893761 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12

Author(s):

Mingkai Zhang ◽

Yang Gao ◽

Jialiang Wang ◽

Zhanbo Liu ◽

Zaishun Jin ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Fluorescence Imaging ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Upconversion Fluorescence ◽

Mantle Cell

In order to determine a particular tumor cell via nanomaterials, we introduce the preparation of CD20 and CD5 coupled nanoprobes (denoted as CD20 and CD5 nanoprobes for convenience) and an application in identification of mantle cell lymphoma (MCL) from B-cell lymphoma. In this work, CD20 and CD5 nanoprobes were prepared by selectively oxidizing the carbon-carbon double bonds of oleate ligands on the surfaces of NaYF4:Yb3+,Tm3+ and NaYF4:Yb3+,Er3+ nanoparticles and, respectively, coupling carboxyl groups on the particles’ surfaces with CD20 and CD5 monoclonal antibodies through EDC/NHS crosslinking agents. After in situ hybridized Jeko-1 cells and Raji cells as a reference with CD20 and CD5 nanoprobes, in vitro double-color upconversion fluorescence imaging of Jeko-1 cells was demonstrated through visualization of blue and green fluorescence under a 980 nm laser excitation. Moreover, in vivo upconversion fluorescence imaging of the transplanted cancer model was also measured. These experimental results indicate that Jeko-1 cells have been specifically labeled by CD20 and CD5 nanoprobes. It is therefore concluded that CD20 and CD5 nanoprobes could be used to specially differentiate mantle cell lymphoma (MCL) from B-cell lymphoma.

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S-phase transcriptional buffering quantified on two different promoters

Life Science Alliance ◽

10.26508/lsa.201800086 ◽

2018 ◽

Vol 1 (5) ◽

pp. e201800086 ◽

Cited By ~ 1

Author(s):

Sharon Yunger ◽

Pinhas Kafri ◽

Liat Rosenfeld ◽

Eliraz Greenberg ◽

Noa Kinor ◽

...

Keyword(s):

Cell Cycle ◽

Transcriptional Activity ◽

Single Cells ◽

Single Gene ◽

Cell System ◽

S Phase ◽

Mrna Transcription ◽

A Cell ◽

Quantitative Fluorescence

Imaging of transcription by quantitative fluorescence-based techniques allows the examination of gene expression kinetics in single cells. Using a cell system for the in vivo visualization of mammalian mRNA transcriptional kinetics at single-gene resolution during the cell cycle, we previously demonstrated a reduction in transcription levels after replication. This phenomenon has been described as a homeostasis mechanism that buffers mRNA transcription levels with respect to the cell cycle stage and the number of transcribing alleles. Here, we examined how transcriptional buffering enforced during S phase affects two different promoters, the cytomegalovirus promoter versus the cyclin D1 promoter, that drive the same gene body. We found that global modulation of histone modifications could completely revert the transcription down-regulation imposed during replication. Furthermore, measuring these levels of transcriptional activity in fixed and living cells showed that the transcriptional potential of the genes was significantly higher than actual transcription levels, suggesting that promoters might normally be limited from reaching their full transcriptional potential.

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Resolving fate and transcriptome of hematopoietic stem cell clones

10.1101/2020.03.25.008433 ◽

2020 ◽

Cited By ~ 3

Author(s):

Weike Pei ◽

Fuwei Shang ◽

Xi Wang ◽

Ann-Kathrin Fanti ◽

Alessandro Greco ◽

...

Keyword(s):

Stem Cell ◽

Cell Fate ◽

Hematopoietic Stem Cell ◽

Single Cells ◽

Molecular Evidence ◽

Hematopoietic Stem ◽

Cell Clones ◽

Embryonic Origin

AbstractAdult bone marrow harbors a mosaic of hematopoietic stem cell (HSC) clones of embryonic origin, and recent work suggests that such clones may have coherent lineage fates. To probe under physiological conditions whether HSC clones with different fates are transcriptionally distinct, we developed PolyloxExpress – a Cre recombinase-dependent DNA substrate for in situ barcoding that allows parallel readout of barcodes and transcriptomes in single cells. We describe differentiation-inactive, multilineage and lineage-restricted HSC clones, find that they reside in distinct regions of the transcriptional landscape of hematopoiesis, and identify corresponding gene signatures. All clone types contain proliferating HSCs, indicating that differentiation-inactive HSCs can undergo symmetric self-renewal. Our work establishes an approach for studying determinants of stem cell fate in vivo and provides molecular evidence for fate coherence of HSC clones.

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