UV LED excited time-resolved fluorescence microscope

Many naturally occurring substances are intrinsically fluorescent (autofluorescent) when exited at an appropriate wavelength and emission can occur throughout the visible spectrum. Autofluorescence is typically a short-lived phenomena with a lifetime (t) measured in nanoseconds and this property is exploited in Time-Resolved Fluorescence (TRF) microscopy to enhance detection of labelled pathogens against autofluorescence background. The TRF methods are based on the use of immunofluorescent labels with long fluorescence lifetimes (~600 μs) to ensure that labelled target is viable long after short-lived autofluorescence has faded. Pulsed excitation is used to excite fluorescence from the sample and this is followed by a gate-delay phase to permit decay of short-lived fluorescence. When flashlamps are used as the excitation source, the gate-delay period must be extended (>50 μs) to ensure that light from the decaying plasma has decayed to zero. The extended gate-delay results in a significant loss of fluorescence intensity from the synthetic label and this is avoided with solidstate excitation sources. A high-power (>100 mW) Light Emitting Diodes (LEDS) (λ 365 nm) was substituted for the flashlamp and found to give excellent background suppression and strong label fluorescence compared to flashlamp excitation.