An integrated micromechanical large particle in flow sorter (MILPIS)

Abstract

At present, the major hurdle to widespread deployment of zebrafish embryo and larvae in large-scale drug development projects is lack of enabling high-throughput analytical platforms. In order to spearhead drug discovery with the use of zebrafish as a model, platforms need to integrate automated pre-test sorting of organisms (to ensure quality control and standardization) and their in-test positioning (suitable for high-content imaging) with modules for flexible drug delivery. The major obstacle hampering sorting of millimetre sized particles such as zebrafish embryos on chip-based devices is their substantial diameter (above one millimetre), mass (above one milligram), which both lead to rapid gravitational-induced sedimentation and high inertial forces. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present an innovative design of a micromechanical large particle in-flow sorter (MILPIS) capable of analysing, sorting and dispensing living zebrafish embryos for drug discovery applications. The system consisted of a microfluidic network, revolving micromechanical receptacle actuated by robotic servomotor and opto-electronic sensing module. The prototypes were fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining. Elements of MILPIS were also fabricated in an optically transparent VisiJet resin using 3D stereolithography (SLA) processes (ProJet 7000HD, 3D Systems). The device operation was based on a rapidly revolving miniaturized mechanical receptacle. The latter function was to hold and position individual fish embryos for (i) interrogation, (ii) sorting decision-making and (iii) physical sorting..The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers embedded in the system. Digital oscilloscope were used to distinguish the diffraction signals from IR sensors when both LIVE and DEAD embryos were flow through in the chip. Image process analysis were also used as detection module to track DEAD embryos as it flowed in the channel. ; phone 61 3 992 57157; fax 61 3 992 57110; http://www.rmit.edu.au/staff/donald-wlodkowic

Deep Learning-Enabled Morphometric Analysis for Toxicity Screening Using Zebrafish Larvae

“…A recent attempt by Neukum et al proposed a promising deep learning-based classification system for fertilized embryos from 2- to 16-cell stage with a specificity of 97% . Although an external physical sorting apparatus was indispensable in such systems, the combination of microfluidic chips and a robotic servomotor for sorting and dispensing could be a feasible solution. , …”

“…51 Although an external physical sorting apparatus was indispensable in such systems, the combination of microfluidic chips and a robotic servomotor for sorting and dispensing could be a feasible solution. 52,53 Given the reality of the growing number of chemicals and the concerns about their hazard potentials, the burden on comprehensive toxicity assessment has rendered the traditional manual operation impracticable and inexecutable. The DLMA developed was successful in revealing the phenotypic variations objectively and accurately.…”

Toward High-Throughput Fish Embryo Toxicity Tests in Aquatic Toxicology

“…(B) A micromechanical large particle in-flow sorter (MILPIS) designed to separate between fertilized and nonfertilized eggs flowing inside a microfluidic channel based on their optical transparency using infrared (IR) beam. Reproduced with permission from ref . (C) Automated and high-throughput dechorionation of fish embryos for toxicity tests.…”

“…Recent proof-of-concept aimed at automated sorting of fertilized Zf eggs include a work by Fuad et al on micromechanical large particle in-flow sorter (MILPIS). , The straightforward and miniaturized system was designed to separate between fertilized and nonfertilized eggs, based on their optical transparency, using infrared (IR) emitters and receivers embedded in a flow through millifluidic chip-based device . Importantly, preliminary tests demonstrated no evident impact of sorting in microfluidic environment on viability or development of Zf embryos.…”