Commercial image analysers and the characterization of microscopical images

Bradbury, S.

doi:10.1111/j.1365-2818.1983.tb04246.x

Cited by 23 publications

(5 citation statements)

References 17 publications

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“…This strategy provides an objective means to compare video equipment and to maximize control of dayto-day variables such as lens alignment, lamp stability, and focus. Moreover, it furnishes a means to initiate the development of radiometric and geometric microscopic fluorescence standards and, finally, allows the application of a whole range of image processing and image analysis methodology (22)(23)(24)(25)(26)(46)(47)(48)(49)(50).…”

Section: Discussionmentioning

confidence: 99%

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Benson¹,

Bryan²,

Plant³

et al. 1985

The Journal of Cell Biology

161

114

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Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 ~m 2 areas in the object plane were obtained.To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2-and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 /~M to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3fl-ol > acridine orange > rhodamine-123 > benzo(a)pyrene > fluorescein > tetramethylrhodamine > 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2Os to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23, 24-dinor-5-cholen-22-amine-3fl-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine. The results show that, with conventional illumination and photographic methods, the fluorescence of regions with high rate constants for photobleaching are significantly underrepresented, and can be as much as 20-fold.Fluorescence microscopy is an attractive method to study the dynamics of various cellular processes and to elucidate the internal architecture of individual cells that uses appropriately labeled fluorescent antibodies and proteins. Work by a large

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Section: Discussionmentioning

confidence: 99%

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Benson¹,

Bryan²,

Plant³

et al. 1985

The Journal of Cell Biology

161

114

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“…Photographs taken at the same magnification were used; furthermore, in order to compare similar areas in different nuclei, only those nuclear sections that contained the nucleolus were considered. Since the nucleus contained several structures that may be of the same level of grey, the image must be pre-processed by interactive methods (Bradbury 1983). We took the following steps: (1) margination of the nucleus, because it could not be discriminated from some structures in the cytoplasm on the basis only of the degree of greyness; and (2) exclusion of the nucleolus from the nucleus; the optical density of the nucleolus was, for the most part, the same as that of condensed chromatin.…”

Section: Image Analysismentioning

confidence: 99%

Nuclear phospholipids during the adaptation of human EUE cells to hypertonic stress

Fraschini

Conti

1995

Histochem Cell Biol

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The phospholipid component of interphase nuclei was analysed in EUE cells (an established cell line from embryonic human epithelium) grown in an isotonic culture medium and during the adaptation process to a hypertonic medium, using a highly specific ultracytochemical procedure, viz. labelling with the phospholipase A2 gold-complex. Within the nucleus, the phospholipids were localized in domains involved in different steps of the synthesis and processing of the RNA. These localizations did not vary at the two key steps of the adaptation process to hypertonic medium: short-term treatment (6 h) representing critical shock condition, and long-term growth (5 days) representing the adapted cells under survival conditions. On the contrary, deep changes of the labelling intensity of phospholipids at these sites occurred at the different times of hypertonic treatment and followed the same course as those observed in the ultramorphological patterns of transcription: the chromatin condensation, as evaluated by image analysis, the permanent nucleolar components, the interchromatin and the perichromatin granules. These data endorse the hypothesis that nuclear phospholipids could be involved in different steps of the transcriptional activity. They are indicative of the deep changes occurring in the EUE cells submitted to hypertonic stress.

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“…The use of computer image analysis is increasingly common in various aspects of microscopy with the benefit of a variety of commercial instruments (Bradbury, 1983). Despite accumulating evidence for their value as an analytical tool, the use of image analysers for quantitative microscopy has not been widespread, due largely to the high cost of the machines.…”

Section: Introductionmentioning

confidence: 99%

Microcomputer video image analysis

Jarvis

1988

Journal of Microscopy

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SUMMARY A review of aspects relating to microcomputer video image analysis is presented. The technical limitations of early systems have been surpassed by high performance video digitizers and microcomputers which are now widely marketed. Commercial systems based on these devices are increasingly available. Also, with the programming skills in most laboratories today, software development for specific applications is a practical proposition provided that sufficient expertise in video and computer technology is available. Considerations necessary for effective hardware selection and software design are discussed by example of the application to light microscope densitometry. Attention is drawn to errors which can arise when employing conventional video systems for analytical purposes.

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Commercial image analysers and the characterization of microscopical images

Cited by 23 publications

References 17 publications

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Nuclear phospholipids during the adaptation of human EUE cells to hypertonic stress

Microcomputer video image analysis

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