Generating multiple-pulse bursts for enhanced fluorescence detection.

The signal-to-background ratio is the limiting factor for fluorescence based detection, sensing, and imaging. A typical background signal will include direct scattering of excitation and Raman scattering of the sample as well as autofluorescence from the sample and additives. To improve the signal-to-background ratio, fluorophores of high brightness and/or high concentration of the fluorophores need to be used. Most of the background is instantaneous and short-lived (picosecond to nanosecond time scale), and using long-lived fluorescence probes combined with time-gated detection allows for significant suppression of unwanted background. Unfortunately, this approach requires substantial sacrifice of the probe signal in order to sufficiently filter the background unless the fluorescence lifetime of the probe is very long. However, long lived probes like ruthenium bipyridyl have relatively low brightness compared to probes that have shorter, 10-30 ns fluorescence lifetimes.We recently presented an approach based on bursts of multiple pulses that allowed for high probe signal amplification using long-lived ruthenium based probe (Ru) and an 80 MHz repetition-rate laser excitation. Unfortunately, Ru represents an extreme case for probe lifetime, and a probe with a shorter lifetime of 20 ns will require excitation from a pulsed source with much higher repetition rate to significantly enhance its signal. Such high repetition rates are not possible to generate with most of today's available electronics. In this report we present new approaches to optimize and generate bursts of pulses with high repetition rate within the burst and no need for new or improved electronics. The high repetition rates originate from a low-repetition source and are highly tunable. We demonstrate that a burst of 2-10 pulses spaced 3 ns apart (corresponding to a 'burst repetition rate' of 330 MHz) allows for high signal enhancement of the 20 ns probe over the sub-nanosecond/nanosecond background. Such an approach can be applied for any sensing format, allowing much higher sensitivity for detection. Since the energy of a single pulse is spread over a few pulses in the burst, the fluorophore's photostability also improves.