Bioassay and Biological Characterization, Amgen, Inc., Thousand Oaks, California 91320, USA.
Cytometry A. 2010 Apr;77(4):356-65. doi: 10.1002/cyto.a.20841.
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.
为了推动悬浮阵列技术的 100 plex 极限，我们开发了全自动的方法来获取多色量子点（QD）编码微球的多光谱图像，对它们进行图像分割，根据颜色编码对它们进行分类，并对多重分析进行定量。微球不是用两种颜色和 n 个级别进行编码，而是用 n 种颜色和两个级别（存在或不存在）进行编码，从而将分类问题从模拟转换为数字。通过可调谐滤波器在系统和分析物标签波长的每个 QD 编码物种的峰值荧光波长处获取沉降在微孔底部的多色微球的图像。另一个微球散射光的图像是在激发带宽中捕获的，该带宽用于在多光谱荧光图像中定位微球。在获取的图像中对物体进行分割，并记录每个通道中每个识别微球的荧光，在此基础上通过应用数学模型和分类算法确定每个微球的“颜色代码”。我们的图像分析程序可以以超过 97%的准确率识别和分类微球，并且分析物的 CV 值低于 20%。这些原理验证结果表明，这种快速、小样本量的格式可以实现对特定蛋白质的高度多重定量。
