scholarly journals Groping for Quantitative Digital 3-D Image Analysis: An Approach to Quantitative Fluorescence In Situ Hybridization in Thick Tissue Sections of Prostate Carcinoma

1997 ◽  
Vol 15 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Karsten Rodenacker ◽  
Michaela Aubele ◽  
Peter Hutzler ◽  
P. S. Umesh Adiga
Keyword(s):  
Image Analysis ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Cell Nucleus ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Necessary Condition ◽  
Confocal Laser ◽  
Thin Sections

In molecular pathology numerical chromosome aberrations have been found to be decisive for the prognosis of malignancy in tumours. The existence of such aberrations can be detected by interphase fluorescence in situ hybridization (FISH). The gain or loss of certain base sequences in the desoxyribonucleic acid (DNA) can be estimated by counting the number of FISH signalsper cell nucleus. The quantitative evaluation of such events is a necessary condition for a prospective use in diagnostic pathology. To avoid occlusions of signals, the cell nucleus has to be analyzed in three dimensions. Confocal laser scanning microscopy is the means to obtain series of optical thin sections from fluorescence stained or marked material to fulfill the conditions mentioned above. A graphical user interface (GUI) to a software package for display, inspection, count and (semi‐)automatic analysis of 3‐D images for pathologists is outlined including the underlying methods of 3‐D image interaction and segmentation developed. The preparative methods are briefly described. Main emphasis is given to the methodical questions of computer‐aided analysis of large 3‐D image data sets for pathologists. Several automated analysis steps can be performed for segmentation and succeeding quantification. However tumour material is in contrast to isolated or cultured cells even for visual inspection, a difficult material. For the present a fully automated digital image analysis of 3‐D data is not in sight. A semi‐automatic segmentation method is thus presented here.

Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions

2015 ◽  
Vol 61 (6) ◽  
pp. 417-428 ◽  
Author(s):  
Edith R. Valle ◽  
Gemma Henderson ◽  
Peter H. Janssen ◽  
Faith Cox ◽  
Trevor W. Alexander ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser

In this study, methanogen-specific coenzyme F420autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

scholarly journals Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

2000 ◽  
Vol 66 (8) ◽  
pp. 3608-3615 ◽  
Author(s):  
Hiroyuki Imachi ◽  
Yuji Sekiguchi ◽  
Yoichi Kamagata ◽  
Akiyoshi Ohashi ◽  
Hideki Harada
Keyword(s):  
In Situ Hybridization ◽  
Pure Culture ◽  
Granular Sludge ◽  
Laser Scanning Microscopy ◽  
Upflow Anaerobic Sludge Blanket ◽  
Confocal Laser ◽  
Anaerobic Sludge ◽  
Thin Sections ◽  
Hydrogenotrophic Methanogens

ABSTRACT The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture withM. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genusDesulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.

scholarly journals Enzymatic Permeabilization of the Thecate Dinoflagellate Alexandrium minutum (Dinophyceae) Yields Detection of Intracellularly Associated Bacteria via Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization

2008 ◽  
Vol 74 (7) ◽  
pp. 2244-2247 ◽  
Author(s):  
Lucía Palacios ◽  
Irma Marín
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Alexandrium Minutum ◽  
Associated Bacteria ◽  
Combined Use ◽  
Catalyzed Reporter Deposition

ABSTRACT The enzymatic permeabilization procedure described here allows the detection of intracellular bacteria in the thecate dinoflagellate Alexandrium minutum by using catalyzed reporter deposition-fluorescence in situ hybridization. The combined use of propidium iodide and calcofluor for confocal laser scanning microscopy, together with general and specific fluorescent bacterial probes, demonstrated the intracellular presence of bacteria, including members of the phylum Bacteroidetes.

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