Hostname: page-component-586b7cd67f-dsjbd Total loading time: 0 Render date: 2024-11-22T08:58:46.472Z Has data issue: false hasContentIssue false
  • English
  • Français

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
...Show all authors
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: [email protected]
Get access

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 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

Both authors contributed equally to this study.

References

Albrecht, D.R. & Bargmann, C.I. (2011). High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments. Nat Methods 8(7), 599–605.Google Scholar
Aufderheide, K. (1986). Identification of the basal bodies and kinetodesmal fibers in living cells of Paramecium tetraurelia Sonneborn, 1975 and Paramecium sonneborni Aufderheide, Daggett & Nerad, 1983. J Protozool 33(1), 77–80.Google Scholar
Aufderheide, K., Du, Q. & Fry, E. (1993). Directed positioning of micronuclei in Paramecium tetraurelia with laser tweezers: Absence of detectible damage after manipulation. J Eukaryot Microbiol 40(6), 793–796.Google Scholar
Aufderheide, K.J. (2008). An overview of techniques for immobilizing and viewing living cells. Micron 39(2), 71–76.Google Scholar
Aufderheide, K.J., Daggett, P.M. & Nerad, T.A. (1983). Paramecium sonneborni, n. sp, a new member of the Paramecium aurelia species-complex. J Protozool 30(1), 128–131.Google Scholar
Aufderheide, K.J., Du, Q. & Fry, E.S. (1992). Directed positioning of nuclei in living Paramecium tetraurelia—Use of the laser optical force trap for developmental biology. Dev Genet 13(3), 235–240.Google Scholar
Aufderheide, K.J. & Janetopoulos, C. (2012). Immobilization of living specimens for microscopic observation. In Current Microscopy Contributions to Advances in Science and Technology, Méndez-Vilas A. (Ed.), pp. 833–838. Badajoz, Spain: Formatex Research Center.Google Scholar
Aufderheide, K.J., Rotolo, T.C. & Grimes, G.W. (1999). Analyses of inverted ciliary rows in Paramecium. Combined light and election microscopic observations. Eur J Protistol 35(1), 81–91.Google Scholar
Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2(11), 764–774.Google Scholar
Axelrod, D. (2003). Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol 361, 1–33.Google Scholar
Bretschneider, T., Diez, S., Anderson, K., Heuser, J., Clarke, M., Muller-Taubenberger, A., Kohler, J. & Gerisch, G. (2004). Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr Biol 14(1), 1–10.Google Scholar
Chronis, N., Zimmer, M. & Bargmann, C.I. (2007). Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans . Nat Methods 4(9), 727–731.Google Scholar
Chung, K., Crane, M.M. & Lu, H. (2008). Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans . Nat Methods 5(7), 637–643.CrossRefGoogle ScholarPubMed
Costa, L.T., Terekhov, A., Rajput, D., Hofmeister, W., Jowhar, D., Wright, G. & Janetopoulos, C. (2011). Femtosecond laser machined microfluidic devices for imaging of cells during chemotaxis. J Laser Appl 23, 042001–042006.Google Scholar
Escudero, L.M., Bischoff, M. & Freeman, M. (2007). Myosin II regulates complex cellular arrangement and epithelial architecture in drosophila. Dev Cell 13(5), 717–729.Google Scholar
Fukui, Y., Yumura, S. & Yumura, T.K. (1987). Agar-overlay immunofluorescence: High-resolution studies of cytoskeletal components and their changes during chemotaxis. Methods Cell Biol 28, 347–356.Google Scholar
Jowhar, D., Wright, G., Samson, P.C., Wikswo, J.P. & Janetopoulos, C. (2010). Open access microfluidic device for the study of cell migration during chemotaxis. Integr Biol (Camb) 2(11-12), 648–658.Google Scholar
Ke, K., Hasselbrink, E.F. & Hunt, A.J. (2005). Rapidly prototyped three-dimensional nanofluidic channel networks in glass substrates. Anal Chem 77(16), 5083–5088.Google Scholar
Lockery, S.R., Lawton, K.J., Doll, J.C., Faumont, S., Coulthard, S.M., Thiele, T.R., Chronis, N., McCormick, K.E., Goodman, M.B. & Pruitt, B.L. (2008). Artificial dirt: Microfluidic substrates for nematode neurobiology and behavior. J Neurophysiol 99(6), 3136–3143.Google Scholar
Mannik, J., Driessen, R., Galajda, P., Keymer, J.E. & Dekker, C. (2009). Bacterial growth and motility in sub-micron constrictions. Proc Natl Acad Sci USA 106(35), 14861–14866.Google Scholar
Martindale, D.W., Allis, C.D. & Bruns, P.J. (1982). Conjugation in Tetrahymena thermophila. A temporal analysis of cytological stages. Exp Cell Res 140(1), 227–236.CrossRefGoogle ScholarPubMed
Matsuoka, S., Miyanaga, Y., Yanagida, T. & Ueda, M. (2012). Preparation of an imaging chamber for visualizing single molecules in living Dictyostelium cells. Cold Spring Harb Protoc 2012(3), 346–348.Google ScholarPubMed
McCormick, K.E., Gaertner, B.E., Sottile, M., Phillips, P.C. & Lockery, S.R. (2011). Microfluidic devices for analysis of spatial orientation behaviors in semi-restrained Caenorhabditis elegans . PLoS One 6(10), e25710. Google Scholar
Mondal, S., Ahlawat, S. & Koushika, S.P. (2012). Simple microfluidic devices for in vivo imaging of C. elegans, Drosophila and zebrafish. J Vis Exp (67), doi:10.3791/3780.Google Scholar
Nanney, D.L. & McCoy, J.W. (1976). Characterization of the species of the Tetrahymena pyriformis complex. Trans Am Microsc Soc 95(4), 664–682.Google Scholar
Ng, S.F. & Frankel, J. (1977). 180 degrees rotation of ciliary rows and its morphogenetic implications in Tetrahymena pyriformis . Proc Natl Acad Sci USA 74(3), 1115–1119.CrossRefGoogle ScholarPubMed
Primiceri, E., Chiriaco, M.S., Rinaldi, R. & Maruccio, G. (2013). Cell chips as new tools for cell biology—Results, perspectives and opportunities. Lab Chip 13(19), 3789–3802.Google Scholar
Seale, K., Janetopoulos, C. & Wikswo, J. (2009). Micro-mirrors for nanoscale three-dimensional microscopy. ACS Nano 3(3), 493–497.CrossRefGoogle ScholarPubMed
Seale, K., Reiserer, R., Markov, D., Ges, I., Wright, C., Janetopoulos, C. & Wikswo, J. (2008). Mirrored pyramidal wells for simultaneous multiple vantage point microscopy. J Microsc 232(1), 1–6.Google Scholar
Shi, W.W., Wen, H., Lin, B.C. & Qin, J.H. (2011). Microfluidic platform for the study of Caenorhabditis elegans . Top Curr Chem 304, 323–338.CrossRefGoogle Scholar
Smith, C.J., Watson, J.D., Spencer, W.C., O'Brien, T., Cha, B., Albeg, A., Treinin, M. & Miller, D.M. III. (2010). Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans . Dev Biol 345(1), 18–33.CrossRefGoogle ScholarPubMed
Spoon, D.M. (1978). A new rotary microcompressor. Trans Am Microsc Soc 97(3), 412–416.Google Scholar
Steinert, M. & Heuner, K. (2005). Dictyostelium as host model for pathogenesis. Cell Microbiol 7, 307–314.Google Scholar
Wang, J., Feng, X., Du, W. & Liu, B.F. (2011). Microfluidic worm-chip for in vivo analysis of neuronal activity upon dynamic chemical stimulations. Anal Chim Acta 701(1), 23–28.Google Scholar
Wazawa, T. & Ueda, M. (2005). Total internal reflection fluorescence microscopy in single molecule nanobioscience. Adv Biochem Eng Biotechnol 95, 77–106.Google Scholar
Westendorf, C., Bae, A.J., Erlenkamper, C., Galland, E., Franck, C., Bodenschatz, E. & Beta, C. (2010). Live cell flattening—traditional and novel approaches. PMC Biophys 3(1), 9.Google Scholar
White, Y.V., Li, X.X., Sikorski, Z., Davis, L.M. & Hofmeister, W. (2008). Single-pulse ultrafast-laser machining of high aspect nano-holes at the surface of SiO2 . Opt Express 16(19), 14411–14420.CrossRefGoogle ScholarPubMed
Whitesides, G.M., Ostuni, E., Takayama, S., Jiang, X. & Ingber, D.E. (2001). Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 3, 335–373.Google Scholar
Wright, G.A., Costa, L., Terekhov, A., Jowhar, D., Hofmeister, W. & Janetopoulos, C. (2012). On-chip open microfluidic devices for chemotaxis studies. Microsc Microanal 18(4), 816–828.Google Scholar
Yang, J., Chen, Z., Yang, F., Wang, S. & Hou, F. (2013). A microfluidic device for rapid screening of chemotaxis-defective Caenorhabditis elegans mutants. Biomed Microdevices 15(2), 211–220.CrossRefGoogle ScholarPubMed
Yanik, M.F., Rohde, C.B. & Pardo-Martin, C. (2011). Technologies for micromanipulating, imaging, and phenotyping small invertebrates and vertebrates. Annu Rev Biomed Eng 13, 185–217.CrossRefGoogle ScholarPubMed

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).

Download Yan Supplementary Material(Video)
Video 20.7 MB

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.

Download Yan Supplementary Material(Video)
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.

Download Yan Supplementary Material(Video)
Video 2.1 MB

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.

Download Yan Supplementary Material(Video)
Video 4.1 MB

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.

Download Yan Supplementary Material(Video)
Video 5.2 MB

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.

Download Yan Supplementary Material(Video)
Video 9.3 MB

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.

Download Yan Supplementary Material(Video)
Video 7.9 MB

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.

Download Yan Supplementary Material(Video)
Video 7.6 MB
Supplementary material: PDF

Yan Supplementary Material

Figures

Download Yan Supplementary Material(PDF)
PDF 928.7 KB