Solid-state time-gated luminescence microscope with ultraviolet light-emitting diode excitation and electron-multiplying charge-coupled device detection

Many naturally occurring materials are autofluorescent, a property that can reduce the discriminative ability of fluorescence methods, sometimes to the point where they cannot be usefully applied. Shifting from the spectral to the temporal domain, it is possible to discriminate fluorophores on the basis of their fluorescence decay lifetime. Luminophores with sufficiently long lifetimes can be discriminated from short-lived autofluorescence using time-gated luminescence (TGL). This technique relies upon the application of a brief excitation pulse followed by a resolving period to permit short-lived autofluorescence to decay, after which detection is enabled to capture persistent emission. In our studies, a high-power UV LED was mounted in the filter capsule of an Olympus BX51 microscope to serve as the excitation source. The microscope was fitted with an Andor DV885 electron-multiplying CCD (EM-CCD) camera with the trigger input synchronized to UV LED operation. Giardia lamblia cysts labeled with the europium chelate BHHST were analyzed against an autofluorescent background with the TGL microscope. The EM-CCD camera captured useful TGL images in real time with a single exposure cycle. With 4x frame averaging, images acquired in TGL mode showed a 30-fold improvement in SNR compared with conventional fluorescence microscopy.