scholarly journals A method to estimate the DNA content of whole nuclei from measurements made on thin tissue sections

Cytometry ◽  
1985 ◽  
Vol 6 (3) ◽  
pp. 234-237 ◽  
Author(s):  
M. Bins ◽  
F. Takens
Keyword(s):  
Dna Content ◽  
Tissue Sections

scholarly journals Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D

2005 ◽  
Vol 27 (4) ◽  
pp. 225-230
Author(s):  
Lennert S. Ploeger ◽  
André Huisman ◽  
Jurryt van der Gugten ◽  
Dionne M. van der Giezen ◽  
Jeroen A. M. Beliën ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Confocal Laser Scanning Microscopy ◽  
Dna Content ◽  
Laser Scanning ◽  
Clinical Applications ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Dna Cytometry ◽  
Tissue Sections

Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

scholarly journals Automated imaging cytometry reveals dysplastic indices of colonic serrated adenomas

2020 ◽  
pp. FSO459
Author(s):  
Nicholas S Samel ◽  
Qin Huang ◽  
Hiroshi Mashimo
Keyword(s):  
Dna Content ◽  
Chromosome Instability ◽  
Neoplastic Progression ◽  
Hyperplastic Polyps ◽  
Dna Index ◽  
Tissue Sections ◽  
Automated Imaging ◽  
Imaging Cytometry ◽  
The Mean ◽  
Serrated Adenomas

Aim: Left-sided colonic serrated adenomas (L-SAs) were evaluated for aneuploidy using automated imaging cytometry to quantify DNA content and compared with normal colonic tissues (NCT), tubular adenomas (TA), left-sided hyperplastic polyps (L-HP) and adenocarcinomas. Materials & methods: We used standard paraffin-embedded Feulgen-stained tissue sections. Results: The mean DNA index (DI) of NCT was 0.95, L-HP was 1.08, TA was 1.22, L-SA was 1.11 and adenocarcinomas was 1.46. DI of L-SA was statistically higher than that of NCT, but not statistically different from L-HP. Conclusion: This study demonstrates that DIs correlate with the described neoplastic progression of L-SA, TA and L-SA compared with NCT and suggests that L-SA may be involved in a chromosome instability pathway of neoplastic progression.

scholarly journals EVIDENCE FOR VARIATION IN THE QUANTITY OF DNA AMONG PLASTIDS OF ACETABULARIA

1970 ◽  
Vol 44 (2) ◽  
pp. 361-375 ◽  
Author(s):  
Christopher L. F. Woodcock ◽  
Lawrence Bogorad
Keyword(s):  
Electron Microscopy ◽  
Acridine Orange ◽  
Dna Content ◽  
Actinomycin D ◽  
Acridine Orange Staining ◽  
Tissue Sections ◽  
Unicellular Algae ◽  
Acetabularia Mediterranea

The DNA content of individual plastids of the giant unicellular algae Acetabularia mediterranea, and Polyphysa cliftoni was studied. Four methods were used for localizing DNA: acridine orange staining, radioautography following actinomycin D-3H treatment, electron microscopy of thin tissue sections, and electron microscopy of osomotically disrupted plastids. With each method, DNA was readily detected in 20–35% of plastids, but no DNA was observed in the remaining 65–80%. The results further showed that in those plastids with detectable DNA the amount of DNA present was variable. The sensitivity and reliability of the localization methods are discussed, and the possible implications of these findings are considered.

scholarly journals Image-Based Method to Quantify Decellularization of Tissue Sections

2021 ◽  
Vol 22 (16) ◽  
pp. 8399
Author(s):  
Maria Narciso ◽  
Jorge Otero ◽  
Daniel Navajas ◽  
Ramon Farré ◽  
Isaac Almendros ◽  
...  
Keyword(s):  
Dna Content ◽  
Phase Contrast ◽  
Tissue Characterization ◽  
Dna Quantification ◽  
Local Entropy ◽  
Image Processing Algorithm ◽  
Tissue Sections ◽  
Contrast Image ◽  
Average Fluorescence ◽  
Tissue Digestion

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.

scholarly journals Dynamic Changes in Quantitative Features of Human Gastric Lesions

1997 ◽  
Vol 13 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Ventzeslav Enchev ◽  
Jean Paul Rigaut
Keyword(s):  
Lymph Node ◽  
Chronic Gastritis ◽  
Dna Content ◽  
Lymph Node Metastases ◽  
Normal Mucosa ◽  
Adenomatous Polyps ◽  
Gastric Lesions ◽  
Tissue Sections ◽  
Node Metastases ◽  
Chronic Ulcers

Quantitative DNA, morphometric cellular and nuclear variables were evaluated in surgical biopsies from patients with various gastric lesions: chronic gastritis, chronic ulcers, adenomatous polyps (gastric adenomas), primary carcinomas and their corresponding lymph‐node metastases. Paraffin‐embedded tissue sections were studied by static cytophotometry (plug method), karyometry (measurements with a graduated eyepiece micrometer of the major and minor axes of the elliptic nuclear profiles, and calculation of profile areas), and measurements of cellular profiles (largest and smallest caliper diameters). Tissue lymphocytes from the same slide were used as diploid controls for the DNA evaluations.An increase of both cellular and nuclear dimensions and DNA content was noted in all pathological tissues, as compared to normal mucosa; the highest values are found in primary gastric carcinomas. A progressive pattern increase of cellular and nuclear dimensions and of DNA content was observed through normal to cancerous tissues, chronic gastritis, chronic ulcers and adenomatous polyps (adenomas). Lymph‐node metastases, in our study, had smaller nuclear (cellular) dimensions than primary cancers.

Head Size and DNA Content in Bacteriophage T4

1972 ◽  
Vol 30 ◽  
pp. 200-201
Author(s):  
Fred Eiserling ◽  
A. H. Doermann ◽  
Linde Boehner
Keyword(s):  
Electron Microscopy ◽  
Dna Content ◽  
Bacteriophage T4 ◽  
Head Size ◽  
Virus Particles ◽  
Altered Protein ◽  
A Cell ◽  
Bacterial Viruses ◽  
A Site ◽  
Protein Shell

The control of form or shape inheritance can be approached by studying the morphogenesis of bacterial viruses. Shape variants of bacteriophage T4 with altered protein shell (capsid) size and nucleic acid (DNA) content have been found by electron microscopy, and a mutant (E920g in gene 66) controlling head size has been described. This mutant produces short-headed particles which contain 2/3 the normal DNA content and which are non-viable when only one particle infects a cell (Fig. 1).We report here the isolation of a new mutant (191c) which also appears to be in gene 66 but at a site distinct from E920g. The most striking phenotype of the mutant is the production of about 10% of the phage yield as “giant” virus particles, from 3 to 8 times longer than normal phage (Fig. 2).

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

Discovery of intracellular 2,8 dihydroxyadenine crystals in bovine lymphatic and hepatic cells

1987 ◽  
Vol 45 ◽  
pp. 906-907
Author(s):  
W. E. Rigsby ◽  
D. M. Hinton ◽  
V. J. Hurst ◽  
P. C. McCaskey
Keyword(s):  
Polarized Light ◽  
Paraffin Sections ◽  
Tissue Samples ◽  
Whole Cells ◽  
Tissue Sections ◽  
Hepatic Cells ◽  
Slaughter House ◽  
Mammalian Tissues ◽  
Carbon Coated ◽  
Cellular Components

Crystalline intracellular inclusions are rarely seen in mammalian tissues and are often difficult to positively identify. Lymph node and liver tissue samples were obtained from two cows which had been rejected at the slaughter house due to the abnormal appearance of these organs in the animals. The samples were fixed in formaldehyde and some of the fixed material was embedded in paraffin. Examination of the paraffin sections with polarized light microscopy revealed the presence of numerous crystals in both hepatic and lymph tissue sections. Tissue sections were then deparaffinized in xylene, mounted, carbon coated, and examined in a Phillips 505T SEM equipped with a Tracor Northern X-ray Energy Dispersive Spectroscopy (EDS) system. Crystals were obscured by cellular components and membranes so that EDS spectra were only obtainable from whole cells. Tissue samples which had been fixed but not paraffin-embedded were dehydrated, embedded in Spurrs plastic, and sectioned.

Ultrastructural aspects of olfactory signal transduction and its development

1994 ◽  
Vol 52 ◽  
pp. 142-143
Author(s):  
Bert Ph. M. Menco
Keyword(s):  
Signal Transduction ◽  
Olfactory Receptor ◽  
Receptor Cell ◽  
Freeze Substitution ◽  
Luminal Surface ◽  
Tissue Sections ◽  
Olfactory Receptor Cells ◽  
Receptor Cells ◽  
Olfactory Signal ◽  
Odor Molecule

Vertebrate olfactory receptor cells are specialized neurons that have numerous long tapering cilia. The distal parts of these cilia line the interface between the external odorous environment and the luminal surface of the olfactory epithelium. The length and number of these cilia results in a large surface area that presumably increases the chance that an odor molecule will meet a receptor cell. Advanced methods of cryoprepration and immuno-gold labeling were particularly useful to preserve the delicate ultrastructural and immunocytochemical features of olfactory cilia required for localization of molecules involved in olfactory signal-transduction. We subjected olfactory tissues to freeze-substitution in acetone (unfixed tissues) or methanol (fixed tissues) followed by low temperature embedding in Lowicryl K11M for that purpose. Tissue sections were immunoreacted with several antibodies against proteins that are presumably important in olfactory signal-transduction.

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