Anderson Kurt I, Sanderson Jeremy, Gerwig Silke, Peychl Jan
Beatson Institute for Cancer Research, Glasgow, UK.
Cytometry A. 2006 Aug 1;69(8):920-9. doi: 10.1002/cyto.a.20323.
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 2004;22:1567-1572; Verkhusha and Lukyanov, 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 live 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 nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging.
基因编码荧光团（荧光蛋白，FPs）的强大功能和简易性吸引了众多分子生物学家投身于光学显微镜研究。第一代荧光蛋白存在激发光谱和发射光谱重叠的问题，这限制了它们成对使用（Patterson等人，《细胞科学杂志》2001年；114（第5部分）：837 - 838）。人们开发了统称为线性解混的图像采集和处理技术，以分离在荧光蛋白对成像以及荧光共振能量转移（FRET）中遇到的重叠荧光信号。这些专门技术并非没有潜在缺点，包括对活细胞成像的灵敏度和时间分辨率的限制，以及在非专业人员手中产生伪影的风险。随着新一代红移荧光蛋白的出现（Shaner等人，《自然生物技术》2004年；22:1567 - 1572；Verkhusha和Lukyanov，《自然生物技术》2004年；22:289 - 296），当需要对荧光蛋白进行简单的双通道成像时，仔细选择激发源和发射滤光片就无需进行线性解混。在这里，我们介绍了蔡司LSM 510激光扫描共聚焦显微镜的一种新配置，该配置针对使用标准光电倍增管对绿色荧光蛋白（GFP）与诸如mRFP1和mCherry等光谱变体进行活细胞成像进行了优化。选择了一台2 mW、594 nm的氦氖激光器作为红色荧光蛋白的激发源。这个波长能有效激发上述红色变体，同时在双通道成像过程中不限制GFP发射的检测范围。与在488和543 nm激发GFP和mCherry相比，在488和594 nm激发时，GFP检测的灵敏度大约提高了一倍，并消除了GFP渗漏到mCherry通道的现象。然而，mCherry检测的灵敏度降低了约30%，这表明需要发射峰更长的红色荧光蛋白。对于具有不重叠发射光谱的荧光蛋白进行同步光学分离的实际优势包括简易性、稳健性、降低产生伪影的风险以及在活细胞成像过程中提高灵敏度。
