Analysis of electrophoretic bands in a substrate
A method and system to enhance analysis of electrophoretic bands by overlaying only the pixels of interest. The overlaid pixels are superimposed as a layer above, i.e., in the foreground of, the overlaid image, i.e., in the background. A user employs the superimposed pixels for molecular weight determination and is still able to generate densitometry analysis of the remaining pixels in the overlaid image.
1. A method for improving analysis of electrophoretic bands in a substrate, the method comprising
receiving a first image of the substrate and a second image of the substrate, the first image comprising a plurality of first electrophoretic bands captured by a first visualizing means, and the second image comprising a plurality of second electrophoretic bands captured by a second visualizing means, where the vertical position of each of the plurality of first electrophoretic bands is associated with a predefined molecular weight such that the first image represents a vertical weight scale;
creating a layered composite image where the first image defines a first layer of the layered composite image and the second image defines a second layer of said layered composite image; and
analyzing the layered composite image, where the analyzing step comprises a first sub-step followed by a second sub-step, where the first sub-step includes performing a molecular weight analysis by comparing information between the first image and the second image as it exists in the layered composite image, and
where the second sub-step includes performing a densitometry analysis by comparing information only within the second layer.
2. The method of claim 1 where the creating step further comprises
identifying a plurality of corresponding pixels defined from a subset of pixels from the first layer that correspond to a subset of pixels from the second layer; and
orienting the first layer with the second layer based at least on an alignment of the plurality of corresponding pixels, resulting in creating the layered composite image.
3. The method of claim 2 where the orienting step further comprises
mapping the positions of a first set of pixels to the positions of a second set of pixels, where the first set of pixels substantially defines the first layer, and the second set of pixels substantially defines said second layer, where mapping is performed in accordance with the positions of the plurality of corresponding pixels resulting in a common coordinate system between the first layer and the second layer.
4. The method of claim 1 where the analyzing first sub-step comprises
determining the vertical position of a second electrophoretic band from the second layer;
matching the vertical position of the second electrophoretic band to a substantially equivalent vertical position on the vertical weight scale from the first layer; and
identifying a molecular weight associated with the second electrophoretic band.
5. The method of claim 4 where the analyzing step further comprises
identifying a molecule associated with the molecular weight; and
associating the second electrophoretic band with the identified molecule.
6. The method of claim 5 where the analyzing step further comprises repeating the previous analyzing steps for those second electrophoretic bands not associated with a molecule.
7. The method of claim 6 wherein the repeating step continues until a sufficient number of second electrophoretic bands have been associated with molecules to establish a source of the second electrophoretic bands.
8. The method of claim 1 where the analyzing second sub-step comprises
measuring an individual area for each of the plurality of second electrophoretic bands;
measuring signal intensity for each of the plurality of second bands; and
calculating a percent purity for each band.
9. The method of claim 1 further comprising controlling an imaging device to capture the said first image by a first visualizing means, then to capture the second image by a second visualizing means.
10. A non-transitory computer readable storage medium having data stored therein representing software executable by a computer, the software including instructions to provide improved analysis of electrophoretic bands in a substrate, the storage medium comprising
instructions for receiving a first image of the substrate and a second image of the substrate, where the first image comprises a plurality of first electrophoretic bands captured by a first visualizing means, and the second image comprises a plurality of second electrophoretic bands captured by a second visualizing means,
where the vertical position of each of the plurality of first electrophoretic bands is associated with a predefined molecular weight such that said first image represents a vertical weight scale;
instructions for creating a layered composite image, where the first image defines a first layer of the layered composite image and the second image defines a second layer of the layered composite image; and
instructions for analyzing the layered composite image, where the instructions for analyzing comprise a first set of instructions followed by a second set of instructions,
where the first set of instructions include performing a molecular weight analysis by comparing information between the first image and the second image as it exists in the layered composite image, and
where the second set of instructions include performing a densitometry analysis by comparing information only within the second layer.
11. The non-transitory computer readable storage medium of claim 10 where the instructions for creating a layered composite image further comprise
instructions for identifying a plurality of corresponding pixels defined from a subset of pixels from the first layer that correspond to a subset of pixels from the second layer; and
instructions for orienting the first layer with the second layer based at least on an alignment of the plurality of corresponding pixels, resulting in the creation of the layered composite image.
12. The non-transitory computer readable storage medium of claim 11 where the instructions for orienting the first layer with the second layer further comprise
instructions for mapping the positions of a first set of pixels to the positions of a second set of pixels, where the first set of pixels substantially define the first layer, and the second set of pixels substantially define the second layer, where the mapping is performed in accord with the positions of the plurality of corresponding pixels, resulting in a common coordinate system between the first layer and the second layer.
13. The non-transitory computer readable storage medium of claim 10 where the first set of instructions for analyzing comprise
instructions for determining the vertical position of a second electrophoretic band from the second layer;
instructions for matching the vertical position of the second electrophoretic band to a substantially equivalent vertical position on the vertical weight scale from the first layer; and
instructions for identifying a molecular weight associated with the second electrophoretic band.
14. The non-transitory computer readable storage medium of claim 10 where the second set of instructions for analyzing comprise
instructions for measuring an individual area for each of the plurality of second electrophoretic bands;
instructions for adding each of the individual areas to acquire a total area; and
instructions for calculating a percent of total weight for each of the plurality of second electrophoretic bands, where each calculation takes the individual area for each band and divides that value by the total area for all bands.
15. The non-transitory computer readable storage medium of claim 10 further comprising instructions for controlling an imaging device to capture the first image by the first visualizing means, and instructions to capture the second image by a second visualizing means.