Real-time analysis of multi-laser-beam fluorescence for timed control of laser tweezers in a microfluidic cell-sorting device

We have developed a microfluidic cell sorter for mammalian cells expressing intrinsic fluorescent proteins that enables selection of cells with proteins that have enhanced photophysical properties, such as reduced fluorescence photobleaching and/or reversible dark state conversion. Previous ensemble imaging studies have used an acousto-optic modulator (AOM) to provide millisecond pulsed laser illumination for in vivo assays that distinguish reversible dark-state conversion from irreversible photobleaching. However, in the sorter, cells are hydrodynamically focused into a stream, which flows through a series of 4 or 8 line-focused, continuous, 532 nm laser beams, such that each cell experiences a similar millisecond modulated excitation. The amplitude and timing of the fluorescence response from each of the beams are measured by a red-sensitive photomultiplier and analyzed in real time to separately determine initial fluorescence brightness and photobleaching characteristics. In addition, each cell's flow speed is found from its time of passage through the beams, and if the analysis results are within adjustable limits, a 1064 nm optical trap beam is switched on and moved along an intersecting trajectory at a matching speed, so that the cell becomes deflected by the optical gradient forces towards another exit channel of the microfluidic device. The optical sorting of cells is similar to that demonstrated by others, except that the motion of the trap beam is achieved using a piezo mirror under computer control, rather than an AOM; also, rather than a single-beam brightness measure using a hardwired circuit, a more complex multi-beam analysis is performed in software using the Real-Time module of LabView (National Instruments) on a separate computer to achieve deterministic timing and low latency. The software displays updated statistics of the sort, obtained by counting cells that pass through an extra laser beam in the exit channel. A mixture of cells expressing different proteins was resolved to select those with slowest photobleaching. Cells collected from the instrument were viable and could reproduce.