Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging
Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging
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DOI：
10.2310/7290.2007.00023
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发表时间：
2007-07-01
影响因子：
2.8
通讯作者：
Mahmood, Umar
中科院分区：
文献类型：
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作者：
Tam, Jenny M.;Upadhyay, Rabi;Mahmood, Umar
绿色荧光蛋白(GFP)已被用于细胞跟踪和成像浅表或手术暴露结构中的基因表达。然而，在活体小鼠成像往往受到几个因素的限制，包括散射和深度衰减以及重叠的自发荧光。自发荧光信号的光谱分布与GFP发射光谱分布明显不同。光谱成像的使用允许通过加权已知的纯成分轮廓来分离和量化这些对活体所见的总荧光信号的贡献。使用荧光连续波单激发和发射带通成像(EFI)很难分离相对GFP和自发荧光信号。为了评估这两种方法的检测阈值，裸鼠皮下注射了一系列表达GFP的细胞。对于EFI，采用了优化的激励和发射带通滤波器。由于使用光谱成像从发射信号中分离自体荧光贡献的能力，与EFI系统记录的发射信号中GFP和自体荧光的混合贡献相比，我们实现了细胞检测下限的300倍提高。光谱成像的检测下限为3×10(3)个细胞，而EFI为1×10(6)个细胞。尽管自发荧光对图像堆叠有贡献，但同一图像中细胞数量的100倍动态范围很容易被可视化。最后，光谱成像能够将红色荧光蛋白的信号干扰从GFP图像中分离出来，反之亦然。这些发现证明了该方法在全动物活体应用中检测低水平多个荧光标记物的实用性。
Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 X 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.