Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography (Conference Presentation)
Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography (Conference Presentation)
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使用荧光寿命光学投影断层扫描对活体斑马鱼进行功能成像（会议演示）
DOI：
10.1117/12.2252721
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Andrews N
中科院分区：
文献类型：
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作者：
Andrews N
目前的显微镜技术还不能很好地成像活体动物的荧光。我们提出荧光寿命光学投影断层扫描（FLIM OPT）应用于成像酶活性的活转基因斑马鱼表达Förster共振能量转移（FRET）生物传感器。OPT可以被认为是x射线CT的光学等效。样本旋转360度，以设定的间隔获取图像，并应用反向投影技术重建三维图像。它可以在透射或荧光模式下进行，允许广泛的可视化技术，包括FLIM。因此，OPT与FRET FLIM的结合可以在3D中提供功能信息。OPT的最佳尺寸范围为mm-cm，填补了共聚焦和MRI之间的尺寸差距，也是斑马鱼的尺寸范围，是理想的成像模型。在TraNac（透明突变体）背景下产生表达Caspase 3 FRET生物传感器的转基因斑马鱼，以提供细胞凋亡的实时读数。我们已经证明，使用FLIM OPT，我们可以检测胚胎和成年Tg（Ubi: caspase3生物传感器）斑马鱼中Caspase3活性的变化。137Cs源照射25 Gy诱导细胞凋亡，照射后头部荧光寿命增加，表明生物传感器裂解和Caspase 3活性增加。通过压缩感知和多路成像的发展，我们已经将OPT和FLIM OPT应用于成年斑马鱼，使我们能够快速获取数据集，以便鱼可以恢复和纵向成像。
Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime optical projection tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Förster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.