Shumilov Dmytro, Rich Ryan M, Gryczynski Ignacy, Raut Sangram, Gryczynski Karol, Kimball Joe, Doan Hung, Sørensen Thomas J, Laursen Bo W, Borejdo Julian, Gryczynski Zygmunt
Department of Physics & Astronomy, Texas Christian University, Fort Worth, TX 76129, USA.
Methods Appl Fluoresc. 2014 May 14;2(2):024009. doi: 10.1088/2050-6120/2/2/024009.
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.
信噪比是基于荧光的检测、传感和成像的限制因素。典型的背景信号将包括激发光的直接散射、样品的拉曼散射以及样品和添加剂的自发荧光。为了提高信噪比，需要使用高亮度的荧光团和/或高浓度的荧光团。大多数背景是瞬时的且寿命较短（皮秒到纳秒时间尺度），使用长寿命荧光探针并结合时间门控检测可以显著抑制不需要的背景。不幸的是，这种方法需要大幅牺牲探针信号才能充分过滤背景，除非探针的荧光寿命非常长。然而，与荧光寿命为10 - 30纳秒的较短探针相比，像联吡啶钌这样的长寿命探针亮度相对较低。我们最近提出了一种基于多个脉冲串的方法，该方法使用长寿命的钌基探针（Ru）和80 MHz重复频率的激光激发实现了高探针信号放大。不幸的是，Ru代表了探针寿命的极端情况，对于寿命为20纳秒的较短探针，需要来自重复频率高得多的脉冲源的激发才能显著增强其信号。使用当今大多数可用电子设备无法产生如此高的重复频率。在本报告中，我们提出了新的方法来优化并产生脉冲串，其在脉冲串内具有高重复频率且无需新的或改进的电子设备。高重复频率源自低重复频率源且高度可调谐。我们证明，间隔3纳秒的2 - 10个脉冲的脉冲串（对应于330 MHz的“脉冲串重复频率”）能够实现20纳秒探针相对于亚纳秒/纳秒背景的高信号增强。这种方法可应用于任何传感形式，实现更高的检测灵敏度。由于单个脉冲的能量分散在脉冲串中的几个脉冲上，荧光团的光稳定性也得到了提高。
