The budding yeast Saccharomyces cerevisiae has been commonly used to study the biology of aging and growth rate. Yet it is difficult to image cells long-term due to their mobility to escape the focal plane as well as the rapid growth nature to overcrowd the imaging area. To address this we have developed a microfluidic device that is capable of mechanically restraining cells in a 72 × 72 × 4 μm (L x W x H) elastic trap area for long-term, real-time observation with 6 programmable on-demand flow switches. The design provides continuous dissection of excess daughter cells outside the well-defined trap site to prevent cell congestion in the 3mm x 3mm x 10 μm chamber. In addition, cells can be held for several days in a focused monolayer and continue to replicate throughout their entire lifespan during imaging. Specifically in the 72 hour experiment, we monitored growth of single budding yeast cells at a fixed xyz position under high magnification to form a monolayer culture without overcrowding the chamber. Image analysis tool was used to track and quantify the number of daughter cells a yeast mother cell produced. Software-driven perturbation provided precise media flow to the thriving colony which was verified by introducing two-color fluorescent to measure viability. Together, the resulting dynamic cell dissection in tightly controlled focused area with long-term real-time imaging offers considerable potential for cell analysis.