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

Published online by Cambridge University Press:  21 January 2014

Yingjun Yan ,
Liwei Jiang ,
Karl J. Aufderheide ,
Gus A. Wright ,
Alexander Terekhov ,
Lino Costa ,
Kevin Qin ,
W. Tyler McCleery ,
John J. Fellenstein  and
Alessandro Ustione
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Yingjun Yan
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA
Liwei Jiang
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
Karl J. Aufderheide
Affiliation:
Department of Biology, Texas A&M University, College Station, TX 77843, USA
Gus A. Wright
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
Alexander Terekhov
Affiliation:
Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN 37388, USA
Lino Costa
Affiliation:
Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN 37388, USA
Kevin Qin
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA
W. Tyler McCleery
Affiliation:
Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37232, USA
John J. Fellenstein
Affiliation:
Vanderbilt Machine Shop, Vanderbilt University, Nashville, TN 37232, USA
Alessandro Ustione
Affiliation:
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA
J. Brian Robertson
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
Carl Hirschie Johnson
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA
David W. Piston
Affiliation:
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA
M. Shane Hutson
Affiliation:
Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37232, USA Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37232, USA
John P. Wikswo
Affiliation:
Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37232, USA Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37232, USA Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232, USA
William Hofmeister
Affiliation:
Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN 37388, USA Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37232, USA
Chris Janetopoulos*
Affiliation:
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN 37232, USA
*
*Corresponding author. E-mail: c.janetopoulos@vanderbilt.edu
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Abstract

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.

Keywords

microcompressionmicrofluidicslaser machiningpyramidal wellsParamecium tetraureliaSaccharomyces cerevisiaepolydimethylsiloxane
Type
Techniques, Software, and Instrumentation Development
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 141 - 151
Copyright
Copyright © Microscopy Society of America 2014 

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Footnotes

†

Both authors contributed equally to this study.

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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).

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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.

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Video 5.9 MB

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.

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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.

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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.

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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.

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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.

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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.

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Supplementary material: PDF

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Figures

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