Rich Ryan M, Gryczynski Ignacy, Fudala Rafal, Borejdo Julian, Stankowska Dorota L, Krishnamoorthy Raghu R, Raut Sangram, Maliwal Badri P, Shumilov Dmytro, Doan Hung, Gryczynski Zygmunt
Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA; Department of Cell Biology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Methods. 2014 Mar 15;66(2):292-8. doi: 10.1016/j.ymeth.2013.08.026. Epub 2013 Aug 29.
Applications of fluorescence based imaging techniques for detection in cellular and tissue environments are severely limited by autofluorescence of endogenous components of cells, tissue, and the fixatives used in sample processing. To achieve sufficient signal-to-background ratio, a high concentration of the probe needs to be used which is not always feasible. Since typically autofluorescence is in the nanosecond range, long-lived fluorescence probes in combination with time-gated detection can be used for suppression of unwanted autofluorescence. Unfortunately, this requires the sacrifice of the large portion the probe signal in order to sufficiently filter the background. We report a simple and practical approach to achieve a many-fold increase in the intensity of a long-lived probe without increasing the background fluorescence. Using controllable, well separated bursts of closely spaced laser excitation pulses, we are able to highly increase the fluorescence signal of a long-lived marker over the endogenous fluorescent background and scattering, thereby greatly increasing detection sensitivity. Using a commercially available confocal microscopy system equipped with a laser diode and time correlated single photon counting (TCSPC) detection, we are able to enhance the signal of a long-lived Ruthenium (Ru)-based probe by nearly an order of magnitude. We used 80 MHz bursts of pulses (12.5 ns pulse separation) repeated with a 320 kHz repetition rate as needed to adequately image a dye with a 380 ns lifetime. Just using 10 pulses in the burst increases the Ru signal almost 10-fold without any increase in the background signal.
基于荧光的成像技术在细胞和组织环境中的检测应用受到细胞、组织内源性成分以及样品处理中所用固定剂自发荧光的严重限制。为了获得足够的信噪比，需要使用高浓度的探针，但这并不总是可行的。由于典型的自发荧光处于纳秒范围内，长寿命荧光探针与时间门控检测相结合可用于抑制不需要的自发荧光。不幸的是，这需要牺牲大部分探针信号才能充分滤除背景。我们报告了一种简单实用的方法，可在不增加背景荧光的情况下使长寿命探针的强度提高许多倍。通过使用可控的、间隔良好的紧密间隔激光激发脉冲串，我们能够使长寿命标记物的荧光信号在高于内源性荧光背景和散射的情况下大幅增加，从而大大提高检测灵敏度。使用配备激光二极管和时间相关单光子计数（TCSPC）检测的商用共聚焦显微镜系统，我们能够将基于钌（Ru）的长寿命探针的信号增强近一个数量级。我们根据需要以320 kHz的重复率使用80 MHz的脉冲串（脉冲间隔为12.5 ns）来对寿命为380 ns的染料进行充分成像。在脉冲串中仅使用10个脉冲就能使Ru信号几乎增加10倍，而背景信号没有任何增加。
