全场OCT及其对小鼠早期胚胎发育的形态学研究
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摘要
光学相干层析（Optical coherence tomography，OCT）是基于低相干干涉测量的一种新型的三维成像手段。该技术因具有非侵入式、低损、高分辨等特点已被广泛应用于生物医学领域。而在发育生物学中，哺乳动物早期胚胎发育机制的研究具有重大的理论意义和应用价值，但由于实验条件及相关方法学的局限性，以形态学演化为核心的早期发育模式与胚胎极性的研究，尚存在一些争议问题亟待解决。
针对这一情况，本论文以OCT的一个分支“全场OCT（Full-field OCT，FF-OCT）”技术为基础，建立了一套研究早期胚胎形态学的新方法和新思路，并应用于小鼠早期胚胎发育的形态学研究中，得到了一系列有特色的成果。
本论文首先发展和完善了全场OCT系统，并且突破全场OCT技术以往只能对固定胚胎（失去活性的胚胎）进行有效成像的限制，第一次成功获取了小鼠早期胚胎从受精卵时期到囊胚时期的早期发育典型阶段非荧光标记、三维活体的图像。这些发育阶段都具有鲜明的形态学特征，本论文也对各个时期主要的形态学参量提出了相应的量化方法，并进行了相关的形态学分析。
进一步，本论文尝试了更为深入的工作：利用全场OCT技术探索与研究早期胚胎动态发育过程中形态学参量之间的四维时空关联。针对第一次卵裂事件中存在的种种争议问题，本论文对第一次卵裂前后的胚胎进行了三维活体成像的对比，并研究了受精卵里的第二极体、雌雄两个原核以及第一卵裂面的变化规律。继而通过相应的量化，以统计学的角度分析了这些形态学参量之间的时空演化机制。实验结果表明，第一卵裂面经过第二极体的概率只有27%，而且利用两个原核能够准确预测真实第一卵裂面的概率也只有24%。这些研究说明，第一卵裂面和受精卵时期的相关形态学参量不存在明确的时空关联。
此外，本论文还利用全场OCT技术进行了与活体细胞内纺锤体结构相关的研究，并首次获取了小鼠卵母细胞和受精卵内纺锤体结构的非荧光标记、三维活体的形态学图像，并对其形态学参量进行了量化与分析。
本论文的研究结果充分展现了全场OCT技术在早期胚胎发育机制与纺锤体相关研究中的应用前景。全场OCT技术将为发育生物学领域进一步的深入研究提供强有力的技术手段。
Optical coherence tomography (OCT) is an emerging3D imaging modalitybased on low coherence interferometry. The technique is suitable fornon-invasive, low optical damage and high resolution tomographic visualizationof the internal structure in biological samples. For these reasons, the techniqueof OCT has become a valuable methodology in biological and medical studies.On the other hand, the mechanism of early mammalian embryonic developmentis of great importance in developmental biology. Among the related studies, dueto the limitation of the experimental conditions and the methods, earlypatterning and polarity that takes the morphological evolution as its core, stillremains a controversial issue.
Therefore, in this dissertation, a novel method based on full-field OCT(FF-OCT), which was an extension of OCT, was presented in order to providethe breakthrough and new insights into the controversial issue. Using thismethod, we demonstrated a series of the interesting results.
First, the system of FF-OCT was successfully established and wasimproved a lot for its application to the morphological studies of early mouseembryos. We broke through the limitation that FF-OCT was only capable ofimaging the fixed (or dead) embryos and obtained label-free3D livemorphological images of the mouse embryos at various typical developmentalstages ranged from1-cell stage to blastocyst stage for the first time. Thesestages were all of notable morphological characteristics and a great many spatialfactors that related to these early developmental stages were quantitativelyanalysed.
For further analysis, we extended our work and made some successfulattempts to study the dynamics of developmental processes during early mouseembryonic lives. We focused our imaging on the controversial event of the firstcleavage to investigate3D spatial morphogenetic relationship between the twopronuclei in zygote, the2PB and the first cleavage plane. Coupled withquantitative and statistical study, we demonstrated that only27%of the first realcleavage planes passed through the2PB and only24%of the predicted first cleavage planes that defined by the two pronuclei were in good accordance withthe real cleavage planes. These results suggested that the2PB and the twopronuclei were not convincing spatial cues for the event of the first cleavage.
In addition, FF-OCT was also applied to the spindle related investigations,and the first label-free3D live morphological images of the spindles within theoocytes and the zygotes were obtained with FF-OCT. The spindle relatedmorphological parameters were quantitatively measured as well.
Our studies demonstrate the feasibility of FF-OCT in providing newinsights and potential breakthroughs to the controversial issues of earlymammalian embryonic development. Also, we believe that FF-OCT will offer apowerful methodology for the early embryo and the spindle related studies inmammalian developmental biology.
针对这一情况，本论文以OCT的一个分支“全场OCT（Full-field OCT，FF-OCT）”技术为基础，建立了一套研究早期胚胎形态学的新方法和新思路，并应用于小鼠早期胚胎发育的形态学研究中，得到了一系列有特色的成果。
本论文首先发展和完善了全场OCT系统，并且突破全场OCT技术以往只能对固定胚胎（失去活性的胚胎）进行有效成像的限制，第一次成功获取了小鼠早期胚胎从受精卵时期到囊胚时期的早期发育典型阶段非荧光标记、三维活体的图像。这些发育阶段都具有鲜明的形态学特征，本论文也对各个时期主要的形态学参量提出了相应的量化方法，并进行了相关的形态学分析。
进一步，本论文尝试了更为深入的工作：利用全场OCT技术探索与研究早期胚胎动态发育过程中形态学参量之间的四维时空关联。针对第一次卵裂事件中存在的种种争议问题，本论文对第一次卵裂前后的胚胎进行了三维活体成像的对比，并研究了受精卵里的第二极体、雌雄两个原核以及第一卵裂面的变化规律。继而通过相应的量化，以统计学的角度分析了这些形态学参量之间的时空演化机制。实验结果表明，第一卵裂面经过第二极体的概率只有27%，而且利用两个原核能够准确预测真实第一卵裂面的概率也只有24%。这些研究说明，第一卵裂面和受精卵时期的相关形态学参量不存在明确的时空关联。
此外，本论文还利用全场OCT技术进行了与活体细胞内纺锤体结构相关的研究，并首次获取了小鼠卵母细胞和受精卵内纺锤体结构的非荧光标记、三维活体的形态学图像，并对其形态学参量进行了量化与分析。
本论文的研究结果充分展现了全场OCT技术在早期胚胎发育机制与纺锤体相关研究中的应用前景。全场OCT技术将为发育生物学领域进一步的深入研究提供强有力的技术手段。
Optical coherence tomography (OCT) is an emerging3D imaging modalitybased on low coherence interferometry. The technique is suitable fornon-invasive, low optical damage and high resolution tomographic visualizationof the internal structure in biological samples. For these reasons, the techniqueof OCT has become a valuable methodology in biological and medical studies.On the other hand, the mechanism of early mammalian embryonic developmentis of great importance in developmental biology. Among the related studies, dueto the limitation of the experimental conditions and the methods, earlypatterning and polarity that takes the morphological evolution as its core, stillremains a controversial issue.
Therefore, in this dissertation, a novel method based on full-field OCT(FF-OCT), which was an extension of OCT, was presented in order to providethe breakthrough and new insights into the controversial issue. Using thismethod, we demonstrated a series of the interesting results.
First, the system of FF-OCT was successfully established and wasimproved a lot for its application to the morphological studies of early mouseembryos. We broke through the limitation that FF-OCT was only capable ofimaging the fixed (or dead) embryos and obtained label-free3D livemorphological images of the mouse embryos at various typical developmentalstages ranged from1-cell stage to blastocyst stage for the first time. Thesestages were all of notable morphological characteristics and a great many spatialfactors that related to these early developmental stages were quantitativelyanalysed.
For further analysis, we extended our work and made some successfulattempts to study the dynamics of developmental processes during early mouseembryonic lives. We focused our imaging on the controversial event of the firstcleavage to investigate3D spatial morphogenetic relationship between the twopronuclei in zygote, the2PB and the first cleavage plane. Coupled withquantitative and statistical study, we demonstrated that only27%of the first realcleavage planes passed through the2PB and only24%of the predicted first cleavage planes that defined by the two pronuclei were in good accordance withthe real cleavage planes. These results suggested that the2PB and the twopronuclei were not convincing spatial cues for the event of the first cleavage.
In addition, FF-OCT was also applied to the spindle related investigations,and the first label-free3D live morphological images of the spindles within theoocytes and the zygotes were obtained with FF-OCT. The spindle relatedmorphological parameters were quantitatively measured as well.
Our studies demonstrate the feasibility of FF-OCT in providing newinsights and potential breakthroughs to the controversial issues of earlymammalian embryonic development. Also, we believe that FF-OCT will offer apowerful methodology for the early embryo and the spindle related studies inmammalian developmental biology.
引文
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