Confocal 3-dimensional DNA image cytometry in thick tissue sections

Czader, Magdalena; Liljeborg, Anders; Auer, Gert; Porwit, Anna

doi:10.1002/(sici)1097-0320(19961101)25:3<246::aid-cyto5>3.0.co;2-d

Cited by 21 publications

(21 citation statements)

References 23 publications

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“…The former is based on manually delineating nuclei in sequential and orthogonal 2D slices using a computer graphics device such as a tablet or mouse, relying on the human visual system and expert judgment (2,3,4). Sometimes, enhanced 3D visualization capabilities are employed (56 -58).…”

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A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks

et al. 2003

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Background: Automated segmentation of fluorescentlylabeled cell nuclei in 3D confocal microscope images is essential to many studies involving morphological and functional analysis. A common source of segmentation error is tight clustering of nuclei. There is a compelling need to minimize these errors for constructing highly automated scoring systems. Methods: A combination of two approaches is presented. First, an improved distance transform combining intensity gradients and geometric distance is used for the watershed step. Second, an explicit mathematical model for the anatomic characteristics of cell nuclei such as size and shape measures is incorporated. This model is constructed automatically from the data. Deliberate initial over-segmentation of the image data is performed, followed by statistical model-based merging. A confidence score is computed for each detected nucleus, measuring

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confidence: 99%

A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks

et al. 2003

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“…Individual thin sections of fixed and stained tissue must be viewed and evaluated one at a time. Three-dimensional follicular reconstructions have been performed for morphological assessments, but the work of image layering has been quite laborious, allowing errors to be introduced in the cutting and realigning of the sections (Schotton and White 1989;Pawley 1995;Czader et al 1996). By maintaining the tissue as an intact structure, these types of problems can be obviated.…”

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Confocal Laser Scanning Microscopy of Rat Follicle Development

Zucker

Keshaviah

Price

et al. 2000

J Histochem Cytochem.

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This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-D ) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.

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“…The fluorescent intensity between successive TIFF sections of a cleared limb did not decease greatly with depth of analysis. Therefore, mathematical treatment of the data to adjust for signal attenuation was not necessary (42,43). It should be emphasized that proper sample clearing procedures are essential to observe throughout thick tissue and acquire signals that can be quantified (12)(13)(14)23,24).…”

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confidence: 99%

Quantitative fluorescence of 5‐FU–treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red

2003

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Recent studies have revealed that an orphan receptor gene of the steroid/thyroid hormone nuclear receptor superfamily, the Nurr1 gene, is essential for the neurogenesis and differentiation of dopaminergic neurons in the midbrain of mice. Transgenic mice lacking the Nurr1 gene soon die after birth and are devoid of dopaminergic neurons in the midbrain. Heterozygous mice survive postnatally without obvious locomotor deficits; however, they have increased vulnerability to dopaminergic neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). In view of the importance of dopamine neurotransmission in brain function, we were interested to know if the human homologous gene of murine Nurr1, the NR4A2 gene, may play a role in the pathogenesis of schizophrenia. We systematically sequenced all the exons of the human NR4A2 gene to search for molecular variants in a cohort of Chinese schizophrenic patients from Taiwan. Two molecular variants were identified: a G‐insertion in intron 6 (designated IVS6 + 17∼ + 18insG), and a G‐deletion in the untranslated exon 1 (designated c.‐469delG). The IVS6 + 17∼ + 18insG is a polymorphic one; further case control study, however, did not reveal association of this polymorphism with schizophrenia. The c.‐469delG is a rare variant found in two unrelated patients among 177 schizophrenic patients, but not in 130 nonpsychotic controls. The result suggests that the c.‐469delG and possibly other variants of the NR4A2 gene may be of relevance to the complex factors involved in the pathogenesis of schizophrenia. © 2001 Wiley‐Liss, Inc.

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Confocal 3-dimensional DNA image cytometry in thick tissue sections

Cited by 21 publications

References 23 publications

A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks

A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks

Confocal Laser Scanning Microscopy of Rat Follicle Development

Quantitative fluorescence of 5‐FU–treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red

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