Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell
Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell
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DOI：
10.1046/j.0022-2720.2001.00984.x
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发表时间：
2002-01-01
影响因子：
2
通讯作者：
Periasamy, A
中科院分区：
文献类型：
--
作者：
Elangovan, M;Day, RN;Periasamy, A
蛋白质相互作用的可视化和定量是生物医学成像的一个新趋势。荧光显微镜的发展，加上新的荧光探针如绿色荧光蛋白的开发，使得荧光共振能量转移（FRET）可以用于研究活体标本中的蛋白质相互作用。基于强度的FRET显微术受到光谱渗透和荧光团浓度的限制。荧光寿命成像（FLIM）显微镜和寿命测量是独立的荧光团浓度或激发强度的变化，和FRET和FLIM的组合提供了高的空间（纳米）和时间（纳秒）分辨率。因为仅测量供体荧光团的寿命，所以光谱渗漏在FRET FLIM成像中不是问题。在本文中，我们描述了一个纳秒FRET-FLIM显微镜仪器的发展，以获得时间分辨图像的供体在存在和不存在的受体。开发了软件来处理单指数和双指数衰减的采集图像。在两种不同条件下测量供体寿命使我们能够准确计算相互作用蛋白质之间的距离。我们使用这种方法来量化的二聚化的转录因子CAATT/增强子结合蛋白α在活垂体细胞。在受体存在下的供体分子寿命的单组分和双组分分析证明了相互作用蛋白质之间的距离分布。
Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging, The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity-based FRET microscopy is limited by spectral bleed-through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed-through is not an issue in FRET-FLIM imaging. In this paper we describe the development of a nanosecond FRET-FLIM microscopy instrumentation to acquire the time-resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one- and two-component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.