Understanding Fluorescence Sensitivity: A Key Parameter In IMD-W™ Detection Performance

The IMD-W system evolved from the core technological principles of its predecessor, the IMD-A® system from BioVigilant. Both instruments use 405nm laser-induced scatter and autofluorescence to detect and discriminate microbes on a particle-by-particle basis.

Due to the increased complexity of an optical system for handling water compared to air, signals from particles and microbes are significantly lower and harder to detect in water. This is a key reason why the IMD-W system was developed subsequent to the IMD-A system. To improve specificity and reduce the possibility of detecting interferent particles, the IMD-W system uses two PMT detectors to discriminate particle signals based upon spectral content.

Flow cytometers are a similar class of instruments used to detect scatter and fluorescence of waterborne particles, and there are many vendors of reference particles for these applications. However, flow cytometers almost exclusively detect extrinsic fluorescence that is applied to a sample using stains, reagents, highly-fluorescent conjugate particles, etc. They are not designed to detect the ultra-low levels of autofluorescence from microbes. As such, the fluorescent particles designed for flow cytometer applications are largely inappropriate for an instrument like the IMD-W system, as fluorescent levels from these particles can be ten times, or even 105 times and beyond, the autofluorescence from a microbe. The tradeoffs for this enhanced signal strength are the requirement for sample prep and inability for a system to operate continuously in-situ. Even if such particles do not saturate the fluorescence detectors on an instrument like the IMD-W system, they do little to confirm performance in the intended application. One exception is the 0.8 μm Spherotech Yellow Low Intensity (STY) bead. Not only is the bead size similar to that of microbes, the fluorescence spectrum and intensity when excited with 405nm is on the same order of magnitude as microbes. These features make it particularly attractive as a general reference for size and fluorescence, and underscore its use as a standard calibration bead on the IMD-A system.