Analysis of electrophoretic bands in a substrate

• US 9,552,529 B2
• Filed: 12/01/2015
• Issued: 01/24/2017
• Est. Priority Date: 03/30/2012
• Status: Active Grant

• Current Assignee: Pierce Biotechnology Incorporated (Perspecta, Inc.)
• Original Assignee: Pierce Biotechnology Incorporated (Perspecta, Inc.)
• Inventors: Berry, Rachael Mae, Jarrett, Nikki L., Hommema, Eric Leigh, Feather-Henigan, Kelli D., Hong, Suk J., Webb, Brian Lynn
• Primary Examiner(s): MOTSINGER, SEAN T
• Application Number:
  US14/955,475
• Publication Number:
  US 20160117565A1
• Time in Patent Office: 420 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: G01N 21/8483 Investigating reagent band test-element handling not specific to a test method G01N33/4875; analytical elements specific to chemical analysis of biological material G01N33/52; autometer with reagent band G01N35/04
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G06F 18/00 Pattern recognition
• CPC Class Codes: G06T 11/60 Editing figures and text; Combining figures or text
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 5/50 using two or more images, e.g. averaging or subtraction
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06V 2201/04 Recognition of patterns in DNA microarrays

Abstract

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.

Claims

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 by a molecular weight analysis and a densitometry analysis, where the molecular weight analysis includes performing a molecular weight determination by comparing information between the first image and the second image as it exists in the layered composite image, and where the densitometry analysis includes performing a densitometry determination 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 molecular weight determination 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 step compares information within the first image and comprises

measuring an individual area for each of the plurality of first electrophoretic bands;

measuring signal intensity for each of the plurality of first bands; and

calculating a percent purity for each band.

9. The method of claim 1 where the densitometry determination 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.

10. 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.

11. A non-volatile 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 and a second set of instructions,

where the first set of instructions include performing a molecular weight determination 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 determination by comparing information only within the second layer.

12. The non-volatile computer readable storage medium of claim 11 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.

13. The non-volatile computer readable storage medium of claim 12 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.

14. The non-volatile computer readable storage medium of claim 11 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.

15. The non-volatile computer readable storage medium of claim 11 where the instructions for analyzing are directed to comparing information within the first image and comprises

instructions for measuring an individual area for each of the plurality of first 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 first electrophoretic bands, where each calculation takes the individual area for each band and divides that value by the total area for all bands.

16. The non-volatile computer readable storage medium of claim 11 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.

17. The non-volatile computer readable storage medium of claim 11 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.

18. A system for improving analysis of electrophoretic bands in a substrate, the system comprising

an imaging device adapted to capture a plurality of digital images including a first image of the substrate and a second image of the substrate,

where the imaging device captures the first image by a first visualizing means and immediately thereafter automatically captures the second image by a second visualizing means, where the first image comprises a plurality of first electrophoretic bands and said second image comprises a plurality of second electrophoretic bands,

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;

a computer adapted for communicating with the imaging device, the computer having at least one processor to execute logic instructions associated with at least one computer software program and a memory for storing the logic instructions,

where the memory stores the first image and the second image received from the imaging device,

where at least one computer software program contains instructions to create a layered composite image from the first image and the second 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

where the at least one computer software program contains instructions to analyze the layered composite image, where the instructions for analyzing comprise a first set of instructions and a second set of instructions, where the first set of instructions include performing a molecular weight determination 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 determination by comparing information only within the second layer; and

a user interface adapted to communicate at least one determination.

19. The system of claim 18 where the computer further comprises at least one computer software program for noise reduction, adapted to reduce noise on a single layer.

20. The system of claim 18 where the user interface comprises a visual display for displaying the layered composite image to a user.