A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs
A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs
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DOI：
10.1002/cyto.a.20323
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发表时间：
2006-08-01
期刊：
影响因子：
3.7
通讯作者：
Peychl, Jan
中科院分区：
文献类型：
--
作者：
Anderson, Kurt I.;Sanderson, Jeremy;Peychl, Jan
基因编码的荧光团（荧光蛋白，FP）的强大和简单吸引了许多分子生物学家对光学显微镜。第一代FP的激发光谱和发射光谱重叠，这限制了它们的成对使用（Patterson等人，J Cell Sci 2001;114（Part 5）：837-838）。图像采集和处理技术，统称为线性解混，已开发出分离重叠的荧光信号中遇到的成像FP对，也在FRET这些专门的技术不是没有其潜在的缺点，包括灵敏度和时间分辨率活细胞成像的限制，和风险的伪影在手中的非专业人士。随着新一代红移FP的出现（Shaner等人，Nat Biotechnol 20041-22：1567-1572; Verkhusha和Lukcyanov，Nat Biotechnol 2004;22：289-296），当需要FP的简单双通道成像时，仔细选择激发源和发射滤光器避免了线性解混的需要。在这里，我们介绍了一种新的配置的蔡司LSM 510激光扫描共聚焦显微镜，优化了五个细胞成像的绿色荧光蛋白（GFP）连同光谱变体，如mRFP 1和mCherry使用标准的光电倍增管。选择2 mW、594 nm HeNe激光器作为红色FP的激发源。该波长有效地激发上述红色变体，而不限制同时双通道成像期间GFP发射的检测范围。与GFP和mCherry在488和543 nm处的激发相比，在488和594 nm处的激发使GFP检测的灵敏度大约加倍，并且消除了GFP渗入mCherry通道。然而，mCherry检测的灵敏度降低了30%，表明需要具有更长发射峰的红色FP。具有非重叠发射光谱的FP的同时光学分离的实际优点包括简单性、鲁棒性、降低的伪影风险以及在活细胞成像期间增加的灵敏度。(c)2006年国际分析细胞学学会
The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 20041-22:1567-1572; Verkhusha and Lukcyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for five cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonover-lapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging. (c) 2006 International Society for Analytical Cytology