Microfluidic system for on-chip high-throughput whole-animal sorting and screening at subcellular resolution

Abstract

We report a suite of key microfluidic devices for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize Caenorhabditis elegans in a well defined geometry for screening phenotypic features at subcellular resolution in physiologically active animals. We show an integrated chip containing individually addressable screening-chamber devices for incubation and exposure of individual animals to biochemical compounds and high-resolution time-lapse imaging of many animals on a single chip without the need for anesthesia. We describe a design for delivery of compound libraries in standard multiwell plates to microfluidic devices and also for rapid dispensing of screened animals into multiwell plates. When used in various combinations, these devices will facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond laser microsurgery for large-scale in vivo neural degeneration and regeneration studies.

Fig. 1.

Microfluidic worm-sorter layout and operation. The sorter consists of control channels and valves (gray) that direct the flow of worms in the flow channels in different directions. The valves are labeled with the letters A–F in the layout, and the actuation order of valves is listed in the table. A value of 1 represents an open valve, and a value of 0 represents a closed valve, as illustrated in the lower-left box. The steps taken to sort each worm are as follows: step 1 (clean), the immobilization chamber is cleaned; step 2 (capture), a worm is captured in the chamber by suction via the top channel while the lower suction channels are inactive; step 3 (wash), the chamber is washed to flush any other worms in the chamber (blue line) toward the waste or the circulator; step 4 (isolate), the chamber is isolated from all of the channels; step 5 (immobilize), the worm is released from the top suction channel and is restrained by the lower suction channels; step 6 (collect), the image acquisition and processing are performed, and the worm is either collected or directed to the waste, depending on its phenotype.

Fig. 2.

Immobilization and subcellular imaging using worm sorter. (A) Image of the on-chip sorter described in Fig. 1. (Scale bar: 500 μm.) (B) A single worm is shown trapped by multiple suction channels. A combined white-light and fluorescence image is taken by a cooled CCD camera with 6.5-μm pixels and a 100-ms exposure time through a ×10 magnification, 0.45 N.A. objective lens with (Nikon). mec-4::GFP-expressing touch neurons and their processes are clearly visible. (Scale bar: 10 μm.) (C) The mechanosensory neurons PLML/R and ALML/R (L, left; R, right) are shown. AVM and PVM extend processes along the anterior and posterior half of the worm and contribute to mechanosensation in these regions. The cell bodies are shown as black dots. PVM, posterior ventral mechanosensory; ALM, anterior lateral mechanosensory; AVM, anterior ventral mechanosensory.

Fig. 3.

Microchamber chip for large-scale screening. (A) The chip consists of chambers connected to flow lines in which the flow path is controlled via multiplexed control lines and valves. (Scale bar: 500 μm.) (B) Each chamber can be addressed independently and loaded with compounds. The flow lines can be flushed with a wash buffer through a dedicated line to prevent cross-contamination. (Scale bar: 500 μm.) (C) The same flow lines can also be used to deliver worms. A special microchamber geometry that consists of circularly arranged microposts is used to immobilize the animals quickly in a well defined geometry by applying a flow without using anesthetics. (Scale bar: 100 μm.) (D) High-resolution images can be taken through the glass substrate of the chip. The GFP-labeled fluorescent touch-neuron image was taken with a white-light background to show a micropost. (Scale bar: 25 μm.) (A–C) Images were taken using a stereomicroscope after loading fluidic lines with color dyes. (D) Image was taken in another setup using an inverted fluorescence microscope.

Fig. 4.

Design for delivery of compounds from standard multiwell plates to microfluidic devices. A microfluidic chip loads compounds from multiwell plates to flow channels by aspiration. The flow lines are multiplexed (14) to direct one compound at a time to a single serial output. The direction of flow in the channels is controlled by microfluidic valves as described in Fig. 1. The flow lines are flushed with a wash buffer after loading each compound to prevent cross-contamination. The single serial output of this device can easily be connected to the microchamber screening chip (Fig. 3) for compound delivery. Each microchamber chip is also multiplexed (14) to sort and deliver compounds to individual chambers (Fig. 3).

Fig. 5.

Possible combinations of our microfluidic technologies for large-scale high-throughput assays. (A) High-speed phenotype screens (e.g., after genetic mutagenesis) can be performed at cellular or subcellular resolution by cascading the microfluidic sorter with the multiwell dispenser. (B) Large-scale RNAi/drug screens can be performed by delivering standard multiwell plate libraries to the microfluidic screening chambers via the multiwell interface chips.

Fig. 6.

Femtosecond laser microsurgery of axons. (A) Optical setup for delivery of femtosecond pulses to a specimen through a high-N.A. objective lens. (B) (Upper) Fluorescence images of mec4::GFP-labeled touch neurons (ALMR, ALML, AVM). L and R, left and right. Femtosecond laser microsurgery is performed on the target process with 100-fs, 3-nJ pulses at a repetition rate of 80 MHz. (Lower) Cell bodies and neural processes identified after edge detection and feature extraction. (Scale bar: 5 μm.) Color coding shows individual cell bodies, neural processes, and the laser cut. Feature extraction was performed by thresholding first to identify cellular features and then, combined with a Canny edge detection algorithm, to identify the outline of neural processes. AVM, anterior ventral mechanosensory; ALM, anterior lateral mechanosensory; L, left; R, right.