FluoVolt Staining Compromises Cell Viability and Morphology during Live-Cell Voltage Imaging

Fluorescent voltage-sensitive dyes (VSDs) enable noninvasive, high-throughput optical monitoring of membrane potential in live cells but may introduce unintended experimental effects that complicate data interpretation. To address this, a key challenge is to characterize and minimize staining- and imaging-associated perturbations to optimize their reliable use across different cell types and imaging conditions. In this study, we systematically evaluate the VSD FluoVolt, its photobleaching, and its effects on cell viability, detachment, and morphology in cancer cell lines (GIN31 and SK-MEL-30) and primary human macrophages. Time-course imaging revealed cell-type-dependent photobleaching, with SK-MEL-30 cells exhibiting rapid fluorescence decay during imaging compared with other cell types. FluoVolt staining combined with excitation was associated with significant cell detachment in GIN31 and SK-MEL-30 cells and with reduced viability accompanied by pronounced morphological changes in GIN31 cells, characterized by a shift from an elongated to an amoeboid-like phenotype. Notably, morphological alterations were observed even in the absence of excitation, indicating that FluoVolt staining alone was also associated with measurable changes in cell morphology. Optimization of staining conditions using reduced dye and loading solution concentrations and incubation time significantly reduced these effects while maintaining measurable fluorescence. Collectively, these findings further characterize the effects associated with FluoVolt staining under the experimental conditions tested, highlight their dependence on cell type and imaging conditions, and emphasize the importance of protocol optimization for the reliable application of FluoVolt in live-cell imaging.