Label-free photoacoustic microscopy: a potential tool for the live imaging of blood disorders in zebrafish.

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 LCR-EGFP 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 casper 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 casper 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.