Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons
Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these adv...
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| Format: | Article |
| Language: | en_US |
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Royal Society of Chemistry
2014
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| Online Access: | http://hdl.handle.net/1721.1/91137 |
| _version_ | 1826212386065874944 |
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| author | Scott, Mark A. Wissner-Gross, Zachary D. Yanik, Mehmet Fatih |
| author2 | Harvard University--MIT Division of Health Sciences and Technology |
| author_facet | Harvard University--MIT Division of Health Sciences and Technology Scott, Mark A. Wissner-Gross, Zachary D. Yanik, Mehmet Fatih |
| author_sort | Scott, Mark A. |
| collection | MIT |
| description | Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics. |
| first_indexed | 2024-09-23T15:20:32Z |
| format | Article |
| id | mit-1721.1/91137 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:20:32Z |
| publishDate | 2014 |
| publisher | Royal Society of Chemistry |
| record_format | dspace |
| spelling | mit-1721.1/911372022-10-02T02:20:04Z Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons Scott, Mark A. Wissner-Gross, Zachary D. Yanik, Mehmet Fatih Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science Scott, Mark A. Wissner-Gross, Zachary D. Yanik, Mehmet Fatih Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics. Hertz Foundation (Fellowship) National Institutes of Health (U.S.) (R01 EUREKA Award 1-R01-NS066352) David & Lucile Packard Foundation (Award in Science and Engineering) 2014-10-21T18:33:16Z 2014-10-21T18:33:16Z 2012-05 2011-11 Article http://purl.org/eprint/type/JournalArticle 1473-0197 1473-0189 http://hdl.handle.net/1721.1/91137 Scott, Mark A., Zachary D. Wissner-Gross, and Mehmet Fatih Yanik. “Ultra-Rapid Laser Protein Micropatterning: Screening for Directed Polarization of Single Neurons.” Lab Chip 12, no. 12 (2012): 2265. en_US http://dx.doi.org/10.1039/c2lc21105j Lab on a Chip Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Royal Society of Chemistry PMC |
| spellingShingle | Scott, Mark A. Wissner-Gross, Zachary D. Yanik, Mehmet Fatih Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons |
| title | Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons |
| title_full | Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons |
| title_fullStr | Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons |
| title_full_unstemmed | Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons |
| title_short | Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons |
| title_sort | ultra rapid laser protein micropatterning screening for directed polarization of single neurons |
| url | http://hdl.handle.net/1721.1/91137 |
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