In vivo photoacoustic imaging of a nonfluorescent E2 crimson genetic reporter in mammalian tissues.
In vivo photoacoustic imaging of a nonfluorescent E2 crimson genetic reporter in mammalian tissues.
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哺乳动物组织中非荧光 E2 深红色基因报告基因的体内光声成像。
DOI：
10.1117/1.jbo.25.4.046004
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发表时间：
2020
影响因子：
3.5
通讯作者：
Ogunlade O
中科院分区：
文献类型：
--
作者：
Ogunlade O
意义：绿色荧光蛋白(GFP)样荧光蛋白因其独立于外部酶或辅因子而形成发色团的独特能力而被广泛用作荧光成像中的遗传报告分子。然而，由于它们在天然状态下具有较低的光声信号产生效率，因此它们在哺乳动物组织中用于光声成像还没有被证明。通过将它们改造成非荧光(NF)，它们的PA生成效率得到了提高。目的：通过修饰E2深红色荧光蛋白(FP)，获得一种暗色的类GFP蛋白报告蛋白，以产生高PA信号产生效率的突变体蛋白，用于活体成像。方法：测定FP和工程突变体蛋白溶液的吸光度、荧光和PA幅度谱，以确定PA信号产生效率最高的突变体。然后，利用逆转录病毒载体在LS174T人结直肠肿瘤细胞中稳定表达该突变体NFA和天然FP，并在连续脉冲光照下测试其光稳定性。为了证明在体内PA信号产生的改善，将表达FP和NFA突变体的细胞注射到小鼠皮下，并使用基于Fabry-Perot的PA扫描仪进行成像。结果：E2 Crimson的NF突变体的荧光强度比FP低2个数量级，PA信号的产生效率更高，NFA产生的PA信号大约是FP的3倍。表达NFA突变体的肿瘤细胞提供了足够的图像对比度，可以在强血管对比度的背景下活体显示，而表达FP的细胞没有产生明显的对比度。结论：首次在哺乳动物组织中证明了GFP样蛋白作为PA成像的遗传报告。这是通过一种突变实现的，它使FP变暗，并提高了PA信号的产生效率。所采取的方法表明，GFP样蛋白可能是目前可用于vivoPA成像的遗传报告队列的一个有前途的补充。
Significance:Green-fluorescent protein (GFP)-like fluorescent proteins are used extensively as genetic reporters in fluorescence imaging due to their distinctive ability to form chromophores independent of external enzymes or cofactors. However, their use for photoacoustic (PA) imaging has not been demonstrated in mammalian tissues because they possess low PA signal generation efficiency in their native state. By engineering them to become nonfluorescent (NF), their PA generation efficiency was increased. This enabled the generation ofin vivocontrast in mice, making it possible for GFP-like proteins to be used as PA genetic reporters in mammalian tissues.Aim:The aim was to develop a darkened GFP-like protein reporter by modifying E2 crimson fluorescent protein (FP) in order to generate NF mutant proteins with high PA signal generation efficiency forin vivoimaging.Approach:The absorbance, fluorescence, and PA amplitude spectra of purified protein solutions of the FP and engineered NF mutants were measured in order to identify the mutant with the highest PA signal generation efficiency. This mutant, referred to as NFA, and the native FP were then stably expressed in LS174T human colorectal tumor cells using a retroviral vector and tested for photostability under continuous pulsed illumination. To demonstrate the improvement in PA signal generationin vivo, cells expressing the FP and NFA mutant were injected subcutaneously in mice and imaged using a Fabry–Perot based PA scanner.Results:The NF mutants of E2 crimson exhibited fluorescence that was 2 orders of magnitude lower than the FP and a higher PA signal generation efficiency; the NFA-generated PA signal was approximately three times higher than the FP. Tumor cells expressing the NFA mutant provided sufficient image contrast to be visualizedin vivoagainst a background of strong vascular contrast, whereas the FP-expressing cells did not generate visible contrast.Conclusion:A GFP-like protein has been demonstrated as a genetic reporter for PA imaging in mammalian tissue for the first time. This was achieved by a mutation, which darkened the FP and increased the PA signal generation efficiency. The approach taken suggests that GFP-like proteins could be a promising addition to the current cohort of genetic reporters available forin vivoPA imaging.