Download פרויקט ביולוגיה חישובית

‫– ‪Final project‬‬
‫‪Computational Biology‬‬
‫‪RNA Quantification‬‬
‫מגישים‪:‬‬
‫מיכל סימון‬
‫חיים בן שימול‬
‫בהנחיית‪:‬‬
‫יהודה ברודי‬
‫ד"ר ירון שב‪-‬טל‬
Introduction
Quantification of single molecules is a rather new
method
Introduction
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It is now possible to fluorescently tag different
molecules within the cell.
Fluorescence microscopy makes it possible track
these molecules (movement, interactions,
kinetics etc.)
The data can be analyzed and quantified using
computational tools.
Project goal
Providing an easy to use tool for quantifying RNA
molecules in cell, determining their location and
distribution.
The tool will facilitate the tracing process of
biological activities in cells.
RNA FISH
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Synthesis of a complementary
oligonucleotide.
Covalently link the
oligonucleotide to a fluorescent
molecule.
Hybridization of the probe with
the RNA of interest.
Detection of the labeled probe
using fluorescent microscopy.
RNA FISH
Wide-Field microscopy technique
Wide-Field microscopy technique
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Fluorescent sample is illuminated
with light of the proper wave
length.
The sample will emit light of a
different wave length.
The light will be detected by a
CCD camera.
The camera will acquire a two
dimensional image of the emitted
light intensity.
Acquiring several 2d planes will
build a 3d representation of the
object.
Wide-Field microscopy technique
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The final image is composed of pixels whose
intensities are proportional to the florescence
emitted by the cell at the represented area.
Image analysis
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Similar tool made in USA.
Image analysis
At the beginning…...
Image analysis
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IMARIS – Tool for analyzing images
Wide graphical abilities.
 Embedded link to MATLAB programs.
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Step I – defining spots
Step II – Particles bounding
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Each particle has a local maxima.
Each maxima is surrounded be local minima points.
Step II – Particles bounding
Defining the right boundaries is essential for correct
quantification!
Boundaries too
wide:
Step II – Particles bounding
Defining the right boundaries is essential for correct
quantification!
Boundaries too
narrow:
Step II – Particles bounding
Defining the right boundaries is essential for correct
quantification!
Reasonable
boundaries:
Step III – Calculating number of
molecules in each particle
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Summing the intensity of each
particle.
Using calibration data for
calculating the number of
molecules in each particle.
Coloring each particle with a
color that will indicate the
number of molecules in it.
Calibration curve
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The light output of a single probe is determined
by measuring the TFI (total fluorescent
intensity) of different dilutions of the probe in a
fixed volume.
The TFI is plotted against the number of
fluorochrome molecules to generate the
calibration curve.
The slope is equal to the TFI per fluorochrome
Calibration curve
The final output
Advantages
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Spots definition allows the user to determine which
particles will be considered.
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Displaying the boundaries upon the image allows the
user to verify the boundaries correctness.
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The coloring of the particles is done upon the image.
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The final data may also be exported to an excel file.
Comparison to the existing tool
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Overlapping spots gave the same number of
molecules in both tools.
Spots defined only by the old tool were found to
be noise rather than real molecules.
Comparison to the existing tool
What’s next?
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Special treatment for the transcription site.
Improving the boundaries definition (currently,
all particles are bounded by squares).
Automatic de-convolution of the image.
Thanks
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Dr. Yaron Shav-Tal
Mr. Yehuda Brody