On-Chip Multiple Particle Velocity and Size Measurement Using Single-Shot Two-Wavelength Differential Image Analysis

Precise and quick measurement of samples' flow velocities is essential for cell sorting timing control and reconstruction of acquired image-analyzed data. We have developed a simple technique for single-shot measurement of flow velocities of particles simultaneously in a microfluidic pathway. The speed was calculated from the difference in the particles' elongation in an acquired image that appeared when two wavelengths of light with different irradiation times were applied. We ran microparticles through an imaging flow cytometer and irradiated two wavelengths of light with different irradiation times simultaneously to those particles. The mixture of two wavelengths transmitted lights was divided into two wavelengths, and the images of the same microparticles for each wavelength were acquired in a single shot. We estimated the velocity from the difference of its elongation divided by the difference of irradiation time by comparing these two images. The distribution of polystyrene beads' velocity was parabolic and highest at the center of the flow channel, consistent with the expected velocity distribution of the laminar flow. Applying the calculated velocity, we also restored the accurate shapes and cross-sectional areas of particles in the images, indicating the capability of this simple method for improvement of imaging flow cytometry and cell sorter for diagnostic screening of circulating tumor cells. In this meeting, we describe the data more in detail and discuss the further application of this method.