Abstract
With their optically transparent appearance, zebrafish larvae are readily imaged with optical-resolution photoacoustic (PA) microscopy (OR-PAM). Previous OR-PAM studies have mapped endogenous chromophores (e.g. melanin and hemoglobin) within larvae; however, anatomical features cannot be imaged with OR-PAM alone due to insufficient optical absorption. We have previously reported on the photoacoustic radiometry (PAR) technique, which can be used simultaneously with OR-PAM to generate images dependent upon the optical attenuation properties of a sample. Here we demonstrate application of the duplex PAR/PA technique for label-free imaging of the anatomy and vasculature of zebrafish larvae in vivo at 200 and 400 MHz ultrasound detection frequencies. We then use the technique to assess the effects of anti-angiogenic drugs on the development of the larval vasculature. Our results demonstrate the effectiveness of simultaneous PAR/PA for acquiring anatomical images of optically transparent samples in vivo, and its potential applications in assessing drug efficacy and embryonic development.
Highlights
In recent years, there has been a rapid increase in the use of zebrafish as specimens for biomedical research [1]
We recently reported on a technique, termed photoacoustic radiometry (PAR), that can be used to simultaneously generate PA images as well as optical attenuation images using conventional transmission style optical-resolution photoacoustic microscopy (OR-photoacoustic microscopy (PAM)) [20,21]
At this stage of development, the most prominent vessels in the trunk are the dorsal aorta (DA) and the posterior cardinal vein (PCV), which run horizontally, and the vertical intersegmental vessels (ISV), which merge at the dorsal side of the zebrafish to form the dorsal longitudinal anastomotic vessel (DLAV) [24]
Summary
There has been a rapid increase in the use of zebrafish as specimens for biomedical research [1]. Conventional transmission-mode optical-resolution photoacoustic microscopy (OR-PAM) approaches have been used to image the eye, vascular system, and heart [12] as well as cardio-cerebrovascular development [13] in vivo in pigment-supressed zebrafish larvae. Other techniques, such as spatial resolution-invariant PAM (SIRPAM) have been used to acquire whole-body images of pigmentation in 3 day-postfertilization (dpf) larvae over a lateral resolution-invariant axial range of 1.8 mm [14]. Multiview imaging techniques have been employed in both PAM [15] and photoacoustic mesoscopy [16] setups to generate images with more isotropic resolution; these studies have only been performed with ex vivo specimens
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