Bacskai Brian J, Skoch Jesse, Hickey Gregory A, Allen Racquel, Hyman Bradley T
Massachusetts General Hospital, Department of Neurology/Alzheimer's Disease Research Laboratory, 114 16th Street, Charlestown, Massachusetts 02129, USA.
J Biomed Opt. 2003 Jul;8(3):368-75. doi: 10.1117/1.1584442.
We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.
我们描述了一种与商用激光扫描多光子显微镜结合使用的商用荧光寿命成像显微镜（FLIM）仪器的应用。飞秒脉冲近红外激光是时域荧光寿命测量的理想激发源。通过与x-y扫描仪同步，可以逐像素获取荧光寿命，具有高空间分辨率。对每个像素进行多指数曲线拟合可得到二维荧光共振能量转移（FRET）测量结果，从而能够确定荧光FRET对的接近程度，以及足够接近以发生FRET的FRET对的比例。文中描述了表征该系统的实验，以及对生物系统中FRET测定有价值的常用试剂。构建了CFP和YFP构建体，以证明这对绿色荧光蛋白（GFP）颜色变体之间的FRET。还研究了常用于免疫组织化学的FRET对荧光素和罗丹明的寿命特征。最后，这些荧光团用于证明从转基因小鼠大脑获得的老年斑的空间分辨FRET。这些结果共同表明，FLIM允许使用市售组件在小于10 nm的空间尺度上灵敏地测量蛋白质-蛋白质相互作用。
