Image processing in microsphere arrays
Described herein are methods for registering analytical images comprising obtaining an image of an array, wherein the image depicts fiducials that can be used to register the sequential images of the array by determining the average intensity of the fiducials and then positioning the sequential images such that the average intensity of the fiducials attains either a maximum or minimum value.
1. A method of registering an analytical image of a microsphere array comprising:
a) providing a microsphere array, wherein said microsphere array comprises:
i) a substrate with a surface comprising discrete sites; and
ii) a population of microspheres comprising at least a first and a second subpopulation each comprising a bioactive agent, wherein said population of microspheres is distributed on said surface such that each of said discrete sites contain no more than 1 microsphere;
b) decoding said microsphere array so that the location of each bioactive agent is elucidated;
c) computing a registration grid based on known locations of said bioactive agents from information obtained from step (b);
d) contacting said array with a sample comprising target analytes;
e) acquiring a hybridization intensity image of said microsphere array, wherein said hybridization intensity image comprises a first bright bead type to serve as a fiducial;
f) overlaying the registration grid onto said hybridization intensity image;
g) determining an average intensity for the first bright bead type; and
h) positioning the registration grid such that the average intensity of the first bright bead type is maximal, thereby registering the analytical image of the microsphere array.
2. A method according to claim 1 , wherein said acquiring said hybridization intensity image comprises contacting said microsphere array with a sample forming a hybridization complex.
3. A method according to claim 1 , wherein said hybridization intensity image comprises a population of microspheres randomly distributed on the surface of a substrate at discrete sites, wherein at least one site does not contain a microsphere, and wherein said site without a microsphere serves as a reference point for the alignment of said registration grid.
4. A method according to claim 1 , wherein said substrate is a fiber optic bundle.
5. A method according to claim 4 , wherein the step of aligning the registration grid to said hybridization intensity image comprises:
(i) aligning said registration grid with hybridization intensity image features near a center area of the microsphere array;
(ii) positioning a first minibundle around said center area;
(iii) tiling other minibundles around said positioned first minibundle until an edge of said sub-bundle is reached;
(iv) using a software program to scan along the edges of said sub-bundle;
(v) correctly positioning said registration grid;
(vi) assigning a first number to each minibundle; and
(vii) assigning a second number to each core of each minibundle.
6. A method according to claim 1 , wherein said microspheres are randomly distributed on the surface of said substrate.
7. A method according to claim 1 , wherein said substrate is selected from the group consisting of glass and plastic.
8. A method according to claim 2 wherein said sample comprises protein.
9. A method according to claim 2 wherein said sample comprises nucleic acid.
10. The method of claim 1 , further comprising determining an average intensity of a second bright bead type and positioning the registration grid such that the average intensity of the second bright bead type is maximal.
11. The method of claim 1 , wherein the step of aligning the registration grid with the hybridization intensity image is performed prior to positioning the registration grid.