High-contrast brightfield (HCBF) microscopy has emerged as a strong tool for noninvasive counting of cells in culture. HCBF imaging delivers precise cell growth data and is completely label free rendering it an attractive alternative to common cell counting procedures that often adversely affect cell growth. With computational image analysis, HCBF achieves efficient high-throughput automated workflows, extremely relevant for drug and chemical screens in pharmaceutical, toxicological, and biomedical research. We demonstrate the applicability of HCBF microscopy to count three common cell types (HEK293, Huh7, and primary human dermal fibroblasts) with diverse morphology challenging the method. The three cell types required different analysis settings, and we identified two parameters of the computational image analysis, which after cell-specific optimization significantly improved the cell counting accuracy, namely the lower size limit and the intensity threshold. Three-dimensional (3D) imaging approaches, which have obtained great attention in recent years, were an interesting prospect to combine with HCBF microscopy. We optimized the analysis of two 3D outputs but found 3D HCBF imaging to be inferior to the optimized single-layer HCBF imaging for cell counting. HCBF cell counts were highly linearly correlated with (R² > 0.99) and highly similar (<15% difference) to cell counts obtained through Hoechst staining, over a broad range of densities allowing at least this level of accuracy for two to three cell generations in Huh7 cells and fibroblasts. Counts of HEK293 cells correlated somewhat less. In conclusion, the HCBF cell counting method is excellently suited for cell proliferation assays and cytotoxicity assays.

中文翻译：

---

优化的高对比度明场显微镜应用，用于人类细胞培养物的非侵入性增殖测定。

高对比度明场（HCBF）显微镜已成为无创计数培养细胞的强大工具。HCBF成像可提供精确的细胞生长数据，并且完全没有标签，这使其成为通常对细胞生长产生不利影响的普通细胞计数程序的有吸引力的替代品。通过计算图像分析，HCBF可以实现高效的高通量自动化工作流程，这与药物，毒理学和生物医学研究中的药物和化学筛选极为相关。我们证明了HCBF显微镜的适用性来计数三种常见的细胞类型（HEK293，Huh7和原代人皮肤成纤维细胞），具有挑战性的多种形态。这三种细胞类型需要不同的分析设置，我们确定了计算图像分析的两个参数，经过针对特定单元的优化后，显着提高了细胞计数的准确性，即尺寸下限和强度阈值。近年来受到关注的三维（3D）成像方法与HCBF显微镜相结合是一个有趣的前景。我们优化了两个3D输出的分析，但发现3D HCBF成像不如用于细胞计数的优化单层HCBF成像。HCBF细胞计数与（R 我们优化了两个3D输出的分析，但发现3D HCBF成像不如用于细胞计数的优化单层HCBF成像。HCBF细胞计数与（R 我们优化了两个3D输出的分析，但发现3D HCBF成像不如用于细胞计数的优化单层HCBF成像。HCBF细胞计数与（R² > 0.99），并且与通过Hoechst染色获得的细胞计数高度相似（相差小于15％），且密度范围很广，从而使Huh7细胞和成纤维细胞中的2至3个细胞世代至少具有这一水平的准确度。HEK293细胞计数的相关性较小。总之，HCBF细胞计数方法非常适用于细胞增殖测定和细胞毒性测定。