Quantification of fluorescent signals

Sabine Mai, Ph.D.Manitoba Institute of Cell Biology

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..

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?

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.

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.

Precision of data.

•Individual genes, •protein localization and movement,•chromosome structure and changes, •three-dimensional (3D) localization within theinterphase nucleus.

Spectral Imaging

Qualitative analysis: genomic rearrangements.

Controls:

SKY on primary cells (normal cells).

Additional methods:

M-FISH, painting, FISH, Southern.

Three-dimensional analysis of the interphase nucleus.

Measurement of relative positions within the nucleus in m scale.

Trisomy 11.

QuickTime™ and aIntel Indeo® Video 5.0 decompressor

are needed to see this picture.

Controls:

Pirmary cell with normal chromosome 11.

Additional methods:

None.

Chromosome painting.

Measurement of length of duplicated chromosome bands.

Controls:

Normal cells (primary cells) without chromosomalchanges.

Additional methods:

Southern blot

Centromere FISH.

Evaluation of centromere numbers and sizes.

Controls:

Internal control for centromere length

Additional methods:

Cytometry analysis of centromere length by FACS.

Q-FISH.

Measurement of telomere length.

Controls:

Internal control for telomere length

Additional methods:

Southern blot to measure relative telomere length

Cytometry analysis of telomere length by FACS.

Fluorescent immunohistochemistry.

Measurement of protein levels andprotein location.

Controls:

- Antibody controls- Cells that serve as positive and negative controls.

Additional methods:

Western analysis.

Fluorescent immunohistochemistry.

Single cell and population analyses.

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.

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

Please see Kim for the distribution of groups

at different workstations

and computers.