Fluorescent-particle analyzer with timing alignment for analog pulse subtraction of fluorescent pulses arising from different excitation locations

摘要

A flow cytometry system (10) includes a flow tube (14) through which particles marked with different fluorochromes pass from a first location (LC1), illuminated by a red laser (LZ1), to a second location (LC2), illuminated by a blue laser (LZ2). A "red" photodetector (PDR) is optically coupled to detect red fluorescence from both locations. "Yellow" (PDY) and "green" (PDG) photodetectors respectively detect yellow and green fluorescence from the second location, while a "scattered light" detector (PDS) detects scattered light from the second location. During a sample run, the red photodetector can output pulses that correspond primarily to APC fluorochrome at the first location and to PerCP at the second location respectively. A delay device (30) delays the APC pulse relative to the PerCP pulse so that the peaks can be scaled and subtracted in the analog electrical domain to remove APC/PerCP crosstalk. The delay is calibrated using a set of APC tagged cells. Each of these cells generates a red fluorescence electrical pulse while at the first location and a scattered light pulse while at the second location. The average time differential between these two pulses for the set of timing cells determines the target delay. This is compared with the actual delay imposed by the delay device. If the error is greater than 0.5 µS, the imposed delay can be adjusted until the delay is below 0.5 µS, or within the tolerance required for reliable difference pulses to be generated by the pulse subtractor.