Segmentation of microspheres in ultrahigh density multiplexed microsphere-based assays

Mathur, Abhishek; Kelso, David M.

doi:10.1117/12.643170

Cited by 5 publications

(22 citation statements)

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“…As a first step, we corrected luminescent images in all five channels for uneven illumination by utilizing “pseudo‐flood” images (25) corresponding to each wavelength (flattening correction). Pseudo‐flood images were images of densely packed microspheres containing only single QD species.…”

Section: Methodsmentioning

confidence: 99%

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

Mathur¹,

Kelso²

2009

Cytometry Pt A

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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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Section: Methodsmentioning

confidence: 99%

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

Mathur¹,

Kelso²

2009

Cytometry Pt A

Self Cite

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“…Therefore, it is desirable to select the optimal distance that achieves the highest packing density while ensuring negligible interference between the microspheres. Note that relates to the ESR through the object model (2).…”

Section: E Design Parameters and Experimental Variablesmentioning

confidence: 99%

“…We consider the illuminating object model as the sum of shell lights from two neighboring microspheres separated at a distance (2) where , with and as the unknown intensities of the shells around microspheres 1 and 2, respectively. We assume that the intensity of a single shell is constant, and express the shell with inner radius and outer radius as…”

Section: A Measurement Modelmentioning

confidence: 99%

“…The target identification method is error-free and it also simplifies the image analysis. Furthermore, by coding microspheres with different receptors, the microsphere arrays can simultaneously capture, identify, and quantify multiple types of targets, which is impossible with traditional microsphere arrays [2]. For the design of the proposed device [8], we employed the posterior Cramér-Rao bound (PCRB) [9] on the mean-square error (MSE) of the target concentration estimation and to choose the optimal design parameters.…”

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Performance Analysis and Design of Position-Encoded Microsphere Arrays Using the Ziv-Zakai Bound

Sarder

Kotagiri

et al. 2013

IEEE Trans.on Nanobioscience

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Position-encoded microsphere arrays are a promising technology for identifying biological targets and quantifying their concentrations. In this paper we analyze the statistical performance of these arrays in imaging targets at typical low signal-to-noise ratio (SNR) levels. We compute the Ziv-Zakai bound (ZZB) on the errors in estimating the unknown parameters, including the target concentrations. We find the SNR level below which the ZZB provides a more accurate prediction of the error than the posterior Cramér-Rao bound (PCRB), through numerical examples. We further apply the ZZB to select the optimal design parameters of the microsphere array device and investigate the effects of the experimental variables such as microscope point-spread function. An imaging experiment on microspheres with protein targets verifies the optimal design parameters using the ZZB.

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“…[1][2][3][4] These arrays have great potential such as the independent, quantitative, and simultaneous assay of multiple types of targets in small volumes of material and collection of statistically rigorous data from numerous microspheres for each type of target. In the microsphere arrays, the microspheres are functionalized with ligands on their surface that are specific to certain targets.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous detection of multiple biological targets using optimized microfluidic microsphere-trap arrays

Sarder

et al. 2014

J. Micro/Nanolith. MEMS MOEMS

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We propose an analytical framework to build a microfluidic microsphere-trap array device that enables simultaneous, efficient, and accurate screening of multiple biological targets in a single microfluidic channel. By optimizing the traps' geometric parameters, the trap arrays in the channel of the device can immobilize microspheres of different sizes at different regions, obeying hydrodynamically engineered trapping mechanism. Different biomolecules can be captured by the ligands on the surfaces of microspheres of different sizes. They are thus detected according to the microspheres' positions (position encoding), which simplifies screening and avoids target identification errors. To demonstrate the proposition, we build a device for simultaneous detection of two target types by trapping microspheres of two sizes. We evaluate the device performance using finite element fluidic dynamics simulations and microsphere-trapping experiments. These results validate that the device efficiently achieves position encoding of the two-sized microspheres with few fluidic errors, providing the promise to utilize our framework to build devices for simultaneous detection of more targets. We also envision utilizing the device to separate, sort, or enumerate cells, such as circulating tumor cells and blood cells, based on cell size and deformability. Therefore, the device is promising to become a cost-effective and point-of-care miniaturized disease diagnostic tool.

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Segmentation of microspheres in ultrahigh density multiplexed microsphere-based assays

Cited by 5 publications

References 0 publications

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

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

Performance Analysis and Design of Position-Encoded Microsphere Arrays Using the Ziv-Zakai Bound

Simultaneous detection of multiple biological targets using optimized microfluidic microsphere-trap arrays

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