What are the limitations of fluorometric cell viability assays?

There are several limitations associated with fluorometric cell viability assays.

• Fluorescence microscopy relies on the availability of appropriate fluorophores and probes. Dependence on probes limits the observation of novel or unexpected structures lacking suitable labeling options.
• Tissue fixation procedures used in fluorescence microscopy can lead to the loss of certain cellular components, such as lipids and small molecules.
• Photobleaching of fluorophores, which results in the loss of fluorescence, is a common issue during imaging.
• Cell damage may occur from exposure to light and fluorophores
• Excitation light used in fluorescence microscopy, particularly at lower wavelengths, can cause DNA damage and generate reactive oxygen species, potentially affecting cell viability.
• Maintaining optimal atmospheric conditions, including temperature, CO2 concentration, and humidity is crucial for live cell imaging. Evaporation of media due to low atmospheric humidity can alter the volume of media as well as the concentration of compounds within the media over time. 
• Fixing and washing steps in sample preparation can be time-consuming and often result in the loss of certain cellular components such as lipids and small molecules.