Optimized High-Contrast Brightfield Microscopy Application for Noninvasive Proliferation Assays of Human Cell Cultures.

Abstract

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.