An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

White, John; Amos, W. B.; Fordham, M

doi:10.1083/jcb.105.1.41

Cited by 793 publications

(361 citation statements)

References 19 publications

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“…The emission filter was a highpass from 550 nm. We used full excitation power and, in agreement with White et al (66) who used comparable settings, we found no degradation of the image quality due to fluorescence saturation.…”

Section: Methodssupporting

confidence: 85%

Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks

et al. 1991

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A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 Fm deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-BOO (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.Key terms: DNA cytometry, confocal laser microscopy, histology, 3-D There is a considerable interest in DNA cytometry, notably because of the importance of searching for correlations of nuclear DNA content with cancer prognosis in individual patients (e.g., 22, 40, 62).The two methods commonly used a t present for nuclear DNA content analysis, image cytometry (ICM) and flow cytometry (FCM), are not devoid of limitations. Observing the tissue architecture may be important in identifying regions for cytometry and, therefore, the main shortcoming of ICM on smears or imprints and of FCM is obviously that they do not allow the study of DNA contents in situ. Even ICM on histological sections does not allow this, as the tissue structure is observed in projection and the resulting nuclear profiles are usually either incomplete if the section is thin or overlapping if the section is thick enough to include a sufficient proportion of complete nuclei. Finally, preparation procedures for ICM and FCM may produce a selection bias of a given cell type (9) and/or nuclear debris (27).'This paper is an expanded version of a work presented at the

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Section: Methodssupporting

confidence: 85%

Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks

et al. 1991

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“…Recent developments in fluorescence microscopy (e.g., White et al, 1987) and vital staining of intracellular membranes with carbocyanine dyes (Terasaki et al, 1984;Terasaki, 1989; have made it possible to follow ER dynamics in living cells (Terasaki et al, 1984(Terasaki et al, , 1986Allan and Brown, 1988;Kachar and Reese, 1988;Terasaki, 1989Terasaki, , 1990Knebel et al, 1990;McCauley and Hepler, 1990). We have developed a technique based on the spreading of dye molecules through the membranes of the ER after microinjection of an oil droplet saturated with dye (Terasaki and Jaffe, 1991).…”

mentioning

confidence: 99%

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

Speksnijder

Terasaki

Hage

et al. 1993

The Journal of Cell Biology

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Abstract. During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phaltusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiICl6(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. . This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.T nE ER is a key cellular organelle involved in the synthesis of membrane lipids and proteins, secretion, and calcium homeostasis. Morphologically, it is organized as a continuous and extensive network of tubules and cisternae with typical three-way junctions. A number of recent studies have revealed that it is a highly dynamic structure which interacts with a variety of cellular constituents, including microtubules and actin filaments (Terasaki et al.,

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“…Using a pinhole to reject out-of-focus light, the optical sectioning capability and hence axial resolution was dramatically improved over standard widefield microscopes (White et al 1987). Theoretically, a small pinhole also increases lateral resolution up to a factor of 1.4.…”

Section: Developments Toward Extended Resolutionmentioning

confidence: 80%

Widefield fluorescence microscopy with extended resolution

Stemmer

Beck

Fiolka

2008

Histochem Cell Biol

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Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.

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An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

Cited by 793 publications

References 19 publications

Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks

Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

Widefield fluorescence microscopy with extended resolution

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