Fluorescence photograph of stomach cancer tumors in Wistar rats: Computerized analysis

La, Fernandez; Yamamori, Hideo; Inoue, Noboru; Ticona, R.; Okui, K

doi:10.1016/0895-6111(92)90029-9

Cited by 3 publications

(2 citation statements)

References 11 publications

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“…There have been few reports dealing with the problem of autofluorescence in fluorescent images. Acquiring baseline autofluorescent images prior to the staining of tissue sections has been used to correct for autofluorescence (4,5). However, this approach is not as useful in FISH because of changes occurring during the denaturation and hybridization procedures.…”

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confidence: 99%

Autofluorescence correction for fluorescence in situ hybridization

et al. 1995

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Fluorescence in situ hybridization (FISH) analysis is a useful diagnostic tool that is being more and more commonly applied to a wide variety of tumor specimens. Application of the FISH technique on touch imprint preparations or on tissue sections, however, is hindered by high autofluorescence. This is especially true when using locus-specific FISH probes with small target size. In this report, we describe an approach to overcome autofluorescence using image processing of digital images.There have been few reports dealing with the problem of autofluorescence in fluorescent images. Acquiring baseline autofluorescent images prior to the staining of tissue sections has been used to correct for autofluorescence (4,5). However, this approach is not as useful in FISH because of changes occurring during the denaturation and hybridization procedures. Quenching of autofluorescence has also been used (3), but this will affect weak specific signals as well. Fluorescence recovery after photobleaching can be used on a pixel-by-pixel basis for autofluorescence correction ( 8 ) , although this approach is cumbersome and requires sophisticated time-resolving equipment. Finally, time-resolved fluorescence of europium chelates can improve the signal-to-noise ratio of fixed samples ( 13). However, this technique also requires a time-resolved microscope and probes that are not readily available. Application of multicolor cornpensation of autofluorescence has not been described for image analysis, although it has been applied on a cell-bycell basis for flow cytometric analysis using single-laser (1,2,10,12) or dual-laser (9,14) excitation.The goal of the present study was to develop a method to increase the sensitivity of detection of cosmid and centromeric probes on tumor touch imprint preparations. The method uses the broad excitation and emission spectra of autofluorescent signals to compensate for autofluorescence in single-colored images. By independently calculating autofluorescence components and Patient MaterialTouch imprint preparations of fresh bladder tumor specimens were prepared by gently touching the slide surface with tumor material. Slides were then air dried and stored under nitrogen at -20°C. DNA Probes and Probe LabelingTwo contiguous erbB-2 cosmid clones (cRCNeu 1 and cRCNeu4), which together span 55 kilobases of genomic DNA (7), were used in combination with a probe specific for the chromosome 17 pericentromeric sequence (p17H8) (15) for two-color FISH analysis. Probes were labeled with fluorescein-1 1 -dUTP or tetramethylrhodamine-1 1 -dUTP for direct labeling and with biotin-1 1 -dUTP or digoxigenin-1 1 -dUTP (Boehringer Mannheim, Indianapolis, IN) for indirect labeling by nick translation using commercially available kits (Bethesda Research Laboratories, Gaithersburg, MD). FISHTouch imprint preparations were fixed in methanol: acetic acid (3:l) and air dried. FISH was performed as described previously ( 1 1 ) with modifications. Briefly, cells on slides were denatured in 70% formamide/2 X SSC ( 1 X SSC is 0....

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confidence: 99%

Autofluorescence correction for fluorescence in situ hybridization

et al. 1995

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“…Several other methods are reported to Overcome the autofluorescence of tissue (Fernandez et al, 1992;Folli et al, 1992). An example is the acquisition of "baseline autofluorescence images" before staining of tissue sections for correction purposes.…”

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confidence: 99%

The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Haas

Verwoerd²,

Corput³

et al. 1996

J Histochem Cytochem.

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The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quantum yield. To increase the intensity of the delayed fluorescence, we have used a recently introduced peroxidase-mediated amplification system. This system results in a large accumulation of biotin-tyramide, which is detected using streptavidin-europium chelate as label. Optimal staining conditions were evaluated for the immunocytochemical detection of vimentin in cryosections of rat liver, for DNA in situ hybridization (alphoid type probes and 40-KB cosmid probes), and for RNA in situ hybridization (detection of 28S ribosomal RNA, human elongation factor mRNA, and luciferase mRNA). Using a time-resolved fluorescence microscope, intense europium fluorescence was obtained in all these applications when the tyramide amplification system was applied. The signals were strong enough to be observed by eye using the microscope in the time-delayed mode. The routine application of this technique for localization and quantization of antigens or nucleic acid sequences in tissue exhibiting strong autofluorescence is discussed.

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Desktop computer-based image analysis of cell surface fluorescence patterning from a photographic source

Latham

Oppenheimer

1996

Acta Histochemica

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Fluorescence photograph of stomach cancer tumors in Wistar rats: Computerized analysis

Cited by 3 publications

References 11 publications

Autofluorescence correction for fluorescence in situ hybridization

Autofluorescence correction for fluorescence in situ hybridization

The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Desktop computer-based image analysis of cell surface fluorescence patterning from a photographic source

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