快速且定量地估计具有各种分布模式的单细胞蛋白仍然是一个技术挑战。此处开发了一种具有均匀光学场（Uni-μFCM）的微流体流式细胞仪，该仪器能够将结合抗体的多色荧光信号转化为具有任意细胞分布的靶向蛋白数量。作为Uni-μFCM的核心，通过采用微加工的金属窗口来塑造激发光束以实现光学激发和荧光检测的均匀光学场，该模型通过数值模拟和实验表征进行验证。在通过测量四个细胞系的可变大小和来源的三种转录因子的单细胞蛋白定量的Uni-μFCM验证之后，Uni-μFCM应用于：（1）量化髓系和淋巴系白血细胞的膜和细胞质标记物以分类细胞系以及正常和患者的血液样本；（2）测量与基因稳定性相关的关键细胞因子的单细胞表达，区分具有不同恶性程度的口腔和结肠肿瘤细胞系，并且（3）对肝肿瘤细胞系、细胞亚型和肝病患者样本进行单细胞干细胞标记物定量，以确定各种谱系等级。通过定量评估复杂的细胞表型，Uni-μFCM大大扩大了单细胞应用程序中关于白血病分类、肿瘤分类和癌症干细胞谱系确定的表型空间。

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-μFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-μFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-μFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-μFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-μFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.