Time-gated luminescence microscopy allowing direct visual inspection of lanthanide-stained microorganisms in background-free condition

Application of standard immuno-fluorescence microscopy techniques for detection of rare-event microorganisms in dirty samples is severely limited by autofluorescence of nontarget organisms or other debris. Time-gated detection using gateable array detectors in combination with microsecond- lifetime luminescent bioprobes (usually lanthanide-based) is highly effective in suppression of (nanosecond-lifetime) autofluorescence background; however, the complexity and cost of the instrumentation is a major barrier to application of these techniques to routine diagnostics. We report a practical, low-cost implementation of time-gated luminescence detection in a standard epifluorescence microscope which has been modified to include a high-power pulsed UV light-emitting diode (LED) illumination source and a standard fast chopper inserted in the focal plane behind amicroscope eyepiece. Synchronization of the pulsed illumination/gated detection cycle is driven from the clock signal fromthe chopper. To achieve time-gated luminescence intensities sufficient for direct visual observation, we use high cycle rates, up to 2.5 kHz, taking advantage of the fast switching capabilities of the LED source. We have demonstrated real-time direct-visual inspection of europium-labeled Giardia lamblia cysts in dirty samples and Cryptosporidium parvum oocysts in fruit juice concentrate. The signal-tobackground ratio has been enhanced by a factor of 18 in time-gatedmode.The availability of low-cost, robust time-gatedmicroscopes will aid development of long-lifetime luminescence bioprobes and accelerate their application in routine laboratory diagnostics.7 page(s