Multispectral image analysis of binary encoded microspheres for highly multiplexed suspension arrays

Abstract

To push the 100-plex envelope of suspension array technology, we have developed fully automated methods to acquire multispectral images of multiplexed quantum-dot (QD) encoded microspheres, to segment them in the images, to classify them based on their color code, and to quantify the multiplexed assays. Instead of coding microspheres with two colors and n levels, microspheres were coded with n colors and two levels (present or absent), thus transforming the classification problem from analog to digital. Images of multiplexed microspheres, sedimented at the bottom of microwells, were acquired through a tunable filter at the peak luminescence wavelength of each QD coding species in the system and the assay label wavelength. Another image of the light scattered from microspheres was captured in the excitation bandwidth that was utilized to localize microspheres in multispectral luminescence images. Objects in the acquired images are segmented and luminescence from each identified microsphere in each channel is recorded, based on which the ''color code'' of each microsphere is determined by applying a mathematical model and a classification algorithm. Our image analysis procedures could identify and classify microspheres with more than 97% accuracy, and the assay CVs were under 20%. These proof-of-principle results demonstrate that highly multiplexed quantification of specific proteins is possible with this rapid, small-sample volume format. ' 2009 International Society for Advancement of Cytometry Key terms multiplexed assays; suspension arrays; microsphere-based arrays; quantum dots; microscopy; image analysis; imaging-based systems; imaging cytometry IN response to emerging needs to perform highly parallel quantification of specific proteins, a number of rapid, low cost, small sample volume analytical methods have been developed (1,2). Planar protein arrays have been designed that can analyze many thousands of proteins in parallel (3,4). They are printed on glass slides and each array element is identified by its location within the array's grid. However, planar arrays require immobilization of each protein on the surface with the same linking chemistry under common conditions, which limits ones ability to optimize epitope presentation and binding site density. Moreover, as each array spot is 100-200 lm in diameter, these arrays are limited to less than 10 4 assays/cm 2 that require relatively large sample volumes for highly multiplexed assays.Microsphere-based suspension arrays were developed to overcome the disadvantages of planar arrays (5-7). Microspheres offer the potential of utilizing different surfaces so each protein can be coated under optimal buffer, pH, and salt concentration conditions. They also allow assays to be performed in suspension thereby accelerating kinetics, reducing sample volumes, and allowing the reaction to proceed under homogeneous conditions. With suspension arrays, microspheres are coated in bulk, producing a large stock of immobilized proteins that can be tested for functi...

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