Deliolanis Nikolaos C, Kasmieh Randa, Wurdinger Thomas, Tannous Bakhos A, Shah Khalid, Ntziachristos Vasilis
Massachusetts General Hospital and Harvard Medical School, Center for Molecular Imaging Research, Laboratory for Bio-Optics and Molecular Imaging, Charlestown, Massachusetts 02139, USA.
J Biomed Opt. 2008 Jul-Aug;13(4):044008. doi: 10.1117/1.2967184.
The discovery of new fluorescent proteins (FPs) that emit in the far-red part of the spectrum, where light absorption from tissue is significantly lower than in the visible, offers the possibility for noninvasive biological interrogation at the entire organ or small animal level in vivo. The performance of FPs in deep-tissue imaging depends not only on their optical characteristics, but also on the wavelength-dependent tissue absorption and the depth of the fluorescence activity. To determine the optimal choice of FP and illumination wavelength, we compared the performance of five of the most promising FPs: tdTomato, mCherry, mRaspberry, mPlum, and Katushka. We experimentally measured the signal strength through mice and employed theoretical predictions to obtain an understanding of the performance of different illumination scenarios, especially as they pertain to tomographic imaging. It was found that the appropriate combination of red-shifted proteins and illumination wavelengths can improve detection sensitivity in small animals by at least two orders of magnitude compared with green FP. It is also shown that the steep attenuation change of the hemoglobin spectrum around the 600-nm range may significantly affect the detection sensitivity and, therefore, necessitates the careful selection of illumination wavelengths for optimal imaging performance.
发现发射光谱处于远红部分的新型荧光蛋白（FPs），在该波段组织对光的吸收明显低于可见光波段，这为在体内对整个器官或小动物进行非侵入性生物学检测提供了可能。荧光蛋白在深层组织成像中的性能不仅取决于其光学特性，还取决于波长依赖性组织吸收以及荧光活性深度。为了确定荧光蛋白和照明波长的最佳选择，我们比较了五种最有前景的荧光蛋白的性能：tdTomato、mCherry、mRaspberry、mPlum和Katushka。我们通过实验测量了透过小鼠的信号强度，并利用理论预测来了解不同照明场景下的性能，特别是与断层成像相关的性能。结果发现，与绿色荧光蛋白相比，红移蛋白和照明波长的适当组合可将小动物的检测灵敏度提高至少两个数量级。研究还表明，血红蛋白光谱在600纳米范围附近的陡峭衰减变化可能会显著影响检测灵敏度，因此，为了获得最佳成像性能，需要仔细选择照明波长。
