The results of this work should provide instrument manufacturers with data, methods, and algorithms for enhancing the clarity and accuracy of information their instruments provide to their customers. Users of these instruments (pharma companies) and regulators, will have better understanding of the factors to consider when comparing results on different types of instruments. In addition, the research may lead to new microfluidic instruments that provide more comprehensive data using smaller sample sizes.
The primary objective is to identify the factors that influence the parameters reported by different measurement techniques. Simultaneous measurement of particle morphology, electrical signal, fluorescence, etc. on each particle produces a data set that will facilitate comparison of results using these techniques separately. In addition, the research will test the capabilities for microfluidics to perform comprehensive subvisible particle analysis on small samples.
A microfluidic device consisting of a microchannel with four electrodes is mounted in a fluorescence/optical microscope. The microscope is outfitted with fast, computer-controlled cameras (both optical and fluorescence) and with a bright 480 nm light emitting diode(LED). The microchannel features an electrical sensing zone, between two electrodes, where the channel width is reduced.
|When a particle enters this zone, the electrical signal triggers the exposure of the cameras. A controlled delay with microsecond resolution is used to trigger the flash of the LED which lasts typically 5 ms. The camera produces an image which is correlated with the electrical signal. Both camera images and electrical signals are stored for each particles analyzed. Fluorescence imaging also makes use of optical filters to isolate fluorescence from labeled particles. By use of multiple flashes during a single exposure, a strobe-like image is obtained of the particle's trajectory through the microchannel. Image-processing software analyzes the particle images for size, aspect ratio, brightness, etc, while peak analysis software analyzes the electrical signals.||
Top: 30 mm long rod in microchannel, middle ESZ signal correlated with image time, inset software-isolated particle used in calculating dimensions, bottom: strobe image of rod tumbling in a microchannel