title: Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability.
abstract: This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries.
authors: Department of Physics, University of Tennessee Knoxville, University of Tennessee Space Institute, Tullahoma, Tennessee 37388, USA. ldavis@utsi.edu
authors: Lloyd M
authors: LM
authors: Davis
authors: Jennifer L
authors: JL
authors: Lubbeck
authors: Kevin M
authors: KM
authors: Dean
authors: Amy E
authors: AE
authors: Palmer
authors: Ralph
authors: R
authors: Jimenez
mesh_terms: D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D038761:Photobleaching; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
references: Nat Methods. 2007 Jan;4(1):81-6
references: 17179937
references: Nat Methods. 2005 Oct;2(10):730
references: 16237856
references: Chem Soc Rev. 2009 Oct;38(10):2823-32
references: 19771329
references: Lab Chip. 2010 Mar 21;10(6):789-94
references: 20221569
references: Anal Chem. 1997 Apr 15;69(8):1564-8
references: 9109354
references: Opt Express. 2004 Sep 20;12(19):4390-8
references: 19483988
references: Lab Chip. 2011 Nov 7;11(21):3656-62
references: 21918752
references: Anal Chem. 2012 May 1;84(9):3929-37
references: 22424298
references: Lab Chip. 2012 Apr 7;12(7):1378-83
references: 22361780
references: Anal Chem. 2008 Aug 15;80(16):6365-72
references: 18510341
references: Nature. 1987 Dec 24-31;330(6150):769-71
references: 3320757
references: Nat Methods. 2008 Jun;5(6):545-51
references: 18454154
references: Biophys J. 2001 May;80(5):2396-408
references: 11325739
references: Biophys J. 2011 Aug 17;101(4):961-9
references: 21843488
references: Nat Methods. 2011 Nov 29;8(12):1019-26
references: 22127219
references: Lab Chip. 2012 Jan 7;12(1):139-45
references: 22072298
references: Annu Rev Phys Chem. 2012;63:595-617
references: 22404588
references: Nat Biotechnol. 1999 Nov;17(11):1109-11
references: 10545919
references: Nat Methods. 2010 Feb;7(2):137-9
references: 20081836
references: Nat Biotechnol. 2005 Jan;23(1):83-7
references: 15608628
references: Lab Chip. 2010 Jun 21;10(12):1567-73
references: 20379604