Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans

Abstract

Microscopy, phenotyping and visual screens are frequently applied to model organisms in combination with genetics. Although widely used, these techniques for multicellular organisms have mostly remained manual and low-throughput. Here we report the complete automation of sample handling, high-resolution microscopy, phenotyping and sorting of Caenorhabditis elegans. The engineered microfluidic system, coupled with customized software, has enabled high-throughput, high-resolution microscopy and sorting with no human intervention and may be combined with any microscopy setup. The microchip is capable of robust local temperature control, self-regulated sample-loading and automatic sample-positioning, while the integrated software performs imaging and classification of worms based on morphological and intensity features. We demonstrate the ability to perform sensitive and quantitative screens based on cellular and subcellular phenotypes with over 95% accuracy per round and a rate of several hundred worms per hour. Screening time can be reduced by orders of magnitude; moreover, screening is completely automated.

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Figure 1: Schematics of the system and the microchip.
Figure 2: Microdevice operation.
Figure 3: Automated analysis of gene-expression pattern in the integrated microsystem.
Figure 4: Automated three-dimensional imaging and sorting with cellular resolution in the integrated microsystem: image processing and decision-making to sort worms based on the number of AWC neurons expressing pstr-2–gfp.
Figure 5: Automated high-throughput microscopy and sorting based on synaptic marker phenotypes.

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Acknowledgements

We acknowledge US National Science Foundation (DBI-0649833) and National Institutes of Health (NS058465) for funding, Caenorhabditis Genetics Center, Y. Jin (University of California San Diego), and C.I. Bargmann (Rockefeller University) for providing strains, H. Brown and Y. Jin for sharing unpublished observations, J. Stirman for technical assistance, and V. Breedveld, R. Butera, T. Streelman and Y. Thio for commenting on the manuscript. M.M.C. is a National Science Foundation graduate fellow.

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Affiliations

Authors

Contributions

K.C., M.M.C. and H.L. designed the experiments. K.C. fabricated the devices, M.M.C. implemented the software, and K.C. and M.M.C. conducted the experiments. K.C., M.M.C. and H.L. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Hang Lu.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-7, Supplementary Methods (PDF 1184 kb)

Supplementary Video 1

No cooling. This movie shows a nematode being imaged at high magnification (100X) without cooling. A pressure gradient is applied to the worm to push it against the side channels in an attempt to keep the animal from moving. As can be easily seen, the worm is still able to move and rotate significantly. (MOV 2199 kb)

Supplementary Video 2

Cooling. This movie shows a nematode being imaged at high magnification (100X) with cooling applied. Unlike Video 1, no pressure gradient is applied to the animal to keep it in place. With cooling alone the animal remains perfectly still for the entire duration of the imaging process. (MOV 2179 kb)

Supplementary Video 3

Routing. This movie shows worms entering the observation chamber and then being routed to either the top or bottom outlet. This demonstrates the routing process of worms during sorting. (MOV 1490 kb)

Supplementary Video 4

Loading regulator. This movie shows the functioning loading regulator. When a worm is positioned inside the observation chamber, the pressure drop across the worm positioned behind the loading regulator is too small to push it into the imaging area. (MOV 1039 kb)

Supplementary Video 5

AWC 1 ON. Representative z-stack of strain CX3695 at 100x on-chip immobilized using cooling showing only one AWC cell body and dendritic/axonal processes. (MOV 498 kb)

Supplementary Video 6

AWC 2 ON. Representative z-stack of strain CX3940 at 100x on-chip immobilized using cooling showing two AWC cell bodies and dendritic/axonal processes. (MOV 736 kb)

Supplementary Video 7

CZ5261. Representative z-stack of strain CZ5261 at 100x on-chip immobilized using cooling. (MOV 648 kb)

Supplementary Video 8

CZ5264. Representative z-stack of strain CZ5264 at 100x on-chip immobilized using cooling. (MOV 1494 kb)

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Chung, K., Crane, M. & Lu, H. Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nat Methods 5, 637–643 (2008). https://doi.org/10.1038/nmeth.1227

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