Immobilization of zebrafish larvae on a chip-based device for environmental scanning electron microscopy (ESEM) imaging

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Laser microsurgery on zebrafish larvae combined with Scanning Electron Microscopy (SEM) imaging can in particular provide accelerated insights into the tissue regeneration phenomena. Conventional SEM exposes, however, specimens to high vacuum environments, and often requires laborintensive and time-consuming pretreatments and manual positioning. Moreover, there are virtually no technologies available that can quickly immobilize the zebrafish larvae for high definition SEM imaging. This work describes the proof-of-concept design and validation of a microfluidic chip-based system for immobilizing zebrafish larvae and it's interfacing with Environmental Scanning Electron Microscope (ESEM) imaging. The Lab-on-a-Chip (LOC) device was fabricated using a high-speed infrared laser micromachining and consists of a reservoir with multiple semispherical microwells, which hold the yolk of zebrafish larvae, and drain channels that allow removing excess of medium during SEM imaging. Paper filter is used to actuate the chip and immobilization of the larvae by gentle suction that occurs during water drainage. The trapping region allows multiple specimens to be positioned on the chip. The device is then inserted directly inside the ESEM and imaged in a near 100% humidity atmosphere. This facilitates ESEM imaging of untreated biological samples.