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A Microfluidic-Enabled Mechanical Microcompressor for the Immobilization of Live Single- and Multi-Cellular Specimens

  • Yingjun Yan (a1) (a2), Liwei Jiang (a1), Karl J. Aufderheide (a3), Gus A. Wright (a1), Alexander Terekhov (a4), Lino Costa (a4), Kevin Qin (a1) (a2), W. Tyler McCleery (a5), John J. Fellenstein (a6), Alessandro Ustione (a7), J. Brian Robertson (a1), Carl Hirschie Johnson (a1), David W. Piston (a7), M. Shane Hutson (a5) (a8), John P. Wikswo (a5) (a7) (a8) (a9), William Hofmeister (a4) (a8) and Chris Janetopoulos (a1) (a2) (a8)...

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

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Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
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Type Description Title
Supplementary Materials

Yan Supplementary Material
P. sonneborni cell immobilized with an Olympus 40 × dry .7 NA lens and imaged with DIC optics. The video is in real time. Note the cilia beating at the periphery of the cell and at the oral apparatus (lower, center part of cell), the trichocyts in the cortical region of the cell, and the large macronucleus just to the left of the vacuole. This movie is acquired at video rate (32 frames/s).

 Video (20.7 MB)
20.7 MB
Supplementary Materials

Yan Supplementary Material
Bright field image of a large field of 5 μm polystyrene beads immobilized by the mechanical microcompressor. The beads in the bottom left are still mobile, while the rest of the field is completely immobilized. In other experiments, we were able to completely immobilize an entire 1 mm × 1 mm field, suggesting that a large area of the coverslip platform could be set and positioned with a defined trapping distance, depending on the size beads used.

 Video (7.6 MB)
7.6 MB
Supplementary Materials

Yan Supplementary Material

 PDF (929 KB)
929 KB
Supplementary Materials

Yan Supplementary Material
Bright field image of 5 μm polystyrene beads compressed into a small “z” volume. Beads were not completely immobilized in this movie. This demonstrates how flat the field becomes as the compressor coverslip begins to interact with the lower coverslip platform. All of the beads seem to be fairly well confined in the same plane. Frames were acquired every 5 s.

 Video (7.9 MB)
7.9 MB
Supplementary Materials

Yan Supplementary Material
Same C. elegans worm as in Supplementary Video 3 and Figures 3c and 3d with newly released embryo. Compression was adjusted to immobilize the embryo. Bright field images were acquired every 5 s.

 Video (4.1 MB)
4.1 MB
Supplementary Materials

Yan Supplementary Material
K. pneumoniae bacterial cells are shown initially trapped by gentle mechanical microcompression and then released. This movie is real time and acquired at video rate (32 frames/s) using a 100 × 1.35 NA lens and imaged with DIC optics.

 Video (5.9 MB)
5.9 MB
Supplementary Materials

Yan Supplementary Material
Phase contrast image of compressed S. cerevisiae cells growing inside a perfusion enabledmechanical compressor. This device had the same manifold as Supplementary Figure 5. The manifold connected to two 1 mm holes drilled in the 12 mm coverslip platform. Yeast grew continuously throughout the 5 h video. Frames acquired every 15 s using a 40 × 1.35 NA lens.

 Video (9.3 MB)
9.3 MB
Supplementary Materials

Yan Supplementary Material
C. elegans worm immobilized on an Olympus upright BH2 microscope with a 40 × 0.65 NA dry lens. Bright field images were acquired every 5 s. Note that the worm is carrying several embryos.

 Video (2.1 MB)
2.1 MB
Supplementary Materials

Yan Supplementary Material
C. elegans worm compressed in a bed of PDMS posts in a perfusion-enabled microcompressor (Supplementary Fig. 6). E. coli expressing GFP were pumped into the device as a food source. Worm is compressed between the posts so that it unable to move laterally and by the compressor coverslip and PDMS floor so it also can’t travel in the z direction. Images were acquired every 1 s.

 Video (5.2 MB)
5.2 MB


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