Quantification of fluorescent signals Sabine Mai, Ph.D. Manitoba Institute of Cell Biology.
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Transcript of Quantification of fluorescent signals Sabine Mai, Ph.D. Manitoba Institute of Cell Biology.
![Page 1: Quantification of fluorescent signals Sabine Mai, Ph.D. Manitoba Institute of Cell Biology.](https://reader036.fdocuments.in/reader036/viewer/2022062516/56649e4e5503460f94b448d7/html5/thumbnails/1.jpg)
Quantification of fluorescent signals
Sabine Mai, Ph.D.Manitoba Institute of Cell Biology
![Page 2: Quantification of fluorescent signals Sabine Mai, Ph.D. Manitoba Institute of Cell Biology.](https://reader036.fdocuments.in/reader036/viewer/2022062516/56649e4e5503460f94b448d7/html5/thumbnails/2.jpg)
Goals:
Reliable analysis of data
Comparison of fluorescent and molecular data
Valid conclusions
Precision of data
Special goals for this workshop:
Rules of quantifications - relationship to other methods - relate this experience to own research program..
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Reliable analysis of data using fluorescence
Qualitative vs.quantitative analysis
FISH signals: how many and where?how intense (deletion vs. amplification)
Protein signals: where? How many?How much protein is expressed?
Controls: what are appropriate controls?
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Comparison of fluorescent and molecular data
FISH: how does it relate to other techniques? How can it be compared? Single cell vs. whole cell population.Precision and combination with other techniques.Morphology.
Protein: how does it relate to other techniques?Protein analysis of individual cells and of populationsProtein localization studies. Precision.Morphology.
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Valid conclusions.
Data can be measured and compared between research andclinical laboratories.
Data are stored and archived and can be revisited any time.
Specific software makes analysis independent of user:valid, reliable, objective.
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Precision of data.
•Individual genes, •protein localization and movement,•chromosome structure and changes, •three-dimensional (3D) localization within theinterphase nucleus.
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Spectral Imaging
Qualitative analysis: genomic rearrangements.
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Controls:
SKY on primary cells (normal cells).
Additional methods:
M-FISH, painting, FISH, Southern.
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Three-dimensional analysis of the interphase nucleus.
Measurement of relative positions within the nucleus in m scale.
Trisomy 11.
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QuickTime™ and aIntel Indeo® Video 5.0 decompressor
are needed to see this picture.
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Controls:
Pirmary cell with normal chromosome 11.
Additional methods:
None.
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Chromosome painting.
Measurement of length of duplicated chromosome bands.
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Controls:
Normal cells (primary cells) without chromosomalchanges.
Additional methods:
Southern blot
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Centromere FISH.
Evaluation of centromere numbers and sizes.
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Controls:
Internal control for centromere length
Additional methods:
Cytometry analysis of centromere length by FACS.
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Q-FISH.
Measurement of telomere length.
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Controls:
Internal control for telomere length
Additional methods:
Southern blot to measure relative telomere length
Cytometry analysis of telomere length by FACS.
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Fluorescent immunohistochemistry.
Measurement of protein levels andprotein location.
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Controls:
- Antibody controls- Cells that serve as positive and negative controls.
Additional methods:
Western analysis.
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Fluorescent immunohistochemistry.
Single cell and population analyses.
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Controls:
- Antibody controls- Cells that serve as positive and negative controls.
Additional methods:
Western analysis.Disadvantages:
- no single cell analysis, only populationanalysis;- simultaneous analysis of several parameters is not possible;- localization of protein(s) is not as obvious.Contaminationissues;- morphology of cell(s) is absent in Western blot.
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Goals for the workshop:
Acquisition of images and analysis on different workstations.Focus today: telomeres, protein, and later 3D analysis.
Analysis/measurements with different software packages:
• Northern Eclipse• Applied Imaging• Teloquant
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Please see Kim for the distribution of groups
at different workstations
and computers.