Microfluidic device for a rapid immobilization of Zebrafish larvae in environmental scanning electron microscopy

Abstract

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold-palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. V C 2014 International Society for Advancement of Cytometry

Review: Microbial analysis in dielectrophoretic microfluidic systems

“…Rather than applying microplate imaging and flow cytometry tools towards single cell analysis, there is great interest in applying label-free microfluidics towards sample manipulation 242 , as well as for trapping single-cells 243,244 , and model organisms such as zebrafish larvae 245 , due to the ability for repeated spatial and temporal analysis of single-cells after delivery of precise drug doses. In this context, DEP-based microfluidic platforms are especially powerful for downstream coupling to imaging technologies, due to their ability to enable rapid, non-invasive and selective immobilization of non-adherent cells, without the need for cumbersome chemistries for surface modification.…”

Sample preparation for flow cytometry benefits from some lateral thinking

“…There is no question that microfluidics, microfabrication, and “lab‐on‐chip” based technologies have the potential to redefine the way we do cytometry. Certainly, it is currently having an impact right across the cytometric landscape . Microfabrication techniques present a method to produce fully enclosed cell sorting systems that overcome issues with both biohazard containment and therapeutic sterility that exist with classical electrostatic droplet sorters.…”