Confocal microscopy for high-resolution and high-content analysis of the cell cycle

Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3-D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high-content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protocol that can bypass such difficulties by an analysisdriven data collection. Cell-cycle analysis of low-resolution data can be employed to select cell populations of interest that can then be imaged at extremely high resolution and subjected to high-content analysis. Curr. Protoc. Cytom. 70:7.42.1-7.42.14.