Light microscopy method offers unique abilities for the determination of membrane transport properties of either single or multiple cells. A stream imaging system composed of a microfluidic device, a charge-coupled device camera, and a microscope has been developed to study the osmotic behavior of multiple cells in response toward their extracellular environment. Cells of interest were first mixed with the desired extracellular medium and streamed into a microchannel. The microchannel confines the movement of the cells in a monolayer and allows cells to move along the flow direction only. The cells then pass through a sensing zone where the images of cells were capable of being captured under a microscope. Using mouse dendritic cells (mDCs) as a model system, the membrane transport properties were investigated. The kinetics volume changes of mDCs under various extracellular conditions at room temperature (22°C) were analyzed using a biophysical model to determine water and cryoprotectant transport properties of the cell membrane. This prototype system directly allows us to observe, trace, capture, and store the sample information in terms of number, concentration, dynamic size, or shape for further analyses and documentations. We believe that the system has the potential of being used as a stand-alone equipment, or integrated into a lab-on-a-chip system, or embedded into commercialized instruments.