Yang Wenzhao, Wang Wei, Jing Lili, Chen Sung-Liang
University of Michigan-Shanghai Jiao Tong University Joint Institute, Shanghai Jiao Tong University, Shanghai 200240, China.
These authors contributed equally to this work.
Biomed Opt Express. 2021 May 26;12(6):3643-3657. doi: 10.1364/BOE.425994. eCollection 2021 Jun 1.
The zebrafish has emerged as a useful model for human hematological disorders. Transgenic zebrafish that express green fluorescence protein (GFP) in red blood cells (RBCs) visualized by fluorescence microscopy (FLM) is a fundamental approach in such studies to understand the cellular processes and biological functions. However, additional and cumbersome efforts are required to breed a transgenic zebrafish line with reliable GFP expression. Further, the yolk autofluorescence and finite GFP fluorescence lifetimes also have an adverse impact on the observation of target signals. Here, we investigate the identification of intracerebral hemorrhage (ICH) and hemolytic anemia (HA) in zebrafish embryos using label-free photoacoustic microscopy (PAM) for imaging. First, ICH and HA in transgenic zebrafish are mainly studied by PAM and FLM. The results show that PAM is comparable to FLM in good identification of ICH and HA. Besides, PAM is more advantageous in circumventing the issue of autofluorescence. Secondly, ICH and HA in the transparent zebrafish without fluorescent labeling are imaged by PAM and bright-field microscopy (BFM). Because of the high contrast to reveal RBCs, PAM obviously outperforms BFM in the identification of both ICH and HA. Note that FLM cannot observe zebrafish due to its lack of fluorescent labeling. Our work proves that PAM can be a useful tool to study blood disorders in zebrafish, which has advantages: (i) Reliable results enabled by intrinsic absorption of RBCs; (ii) wide applicability to zebrafish strains (no requirement of a transgene); (iii) high sensitivity in identification of ICH and HA compared with BFM.
斑马鱼已成为研究人类血液系统疾病的一种有用模型。通过荧光显微镜（FLM）观察，在红细胞（RBC）中表达绿色荧光蛋白（GFP）的转基因斑马鱼是此类研究中了解细胞过程和生物学功能的基本方法。然而，培育具有可靠GFP表达的转基因斑马鱼品系需要额外且繁琐的工作。此外，卵黄自发荧光和有限的GFP荧光寿命也对目标信号的观察产生不利影响。在此，我们研究了使用无标记光声显微镜（PAM）成像来识别斑马鱼胚胎中的脑出血（ICH）和溶血性贫血（HA）。首先，主要通过PAM和FLM研究转基因斑马鱼中的ICH和HA。结果表明，在良好识别ICH和HA方面，PAM与FLM相当。此外，PAM在规避自发荧光问题方面更具优势。其次，通过PAM和明场显微镜（BFM）对无荧光标记的透明斑马鱼中的ICH和HA进行成像。由于在显示RBC方面具有高对比度，PAM在识别ICH和HA方面明显优于BFM。注意，由于缺乏荧光标记，FLM无法观察斑马鱼。我们的工作证明，PAM可以成为研究斑马鱼血液疾病的有用工具，其具有以下优点：（i）由RBC的固有吸收实现可靠结果；（ii）对斑马鱼品系具有广泛适用性（无需转基因）；（iii）与BFM相比，在识别ICH和HA方面具有高灵敏度。
