title: Time-resolved microscopy for imaging lanthanide luminescence in living cells.
abstract: Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (<100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, λ(em) = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). About 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. About 40 nm microspheres containing ~400 chelate molecules were detected within ~1-s acquisition times. A luminescent terbium complex, Lumi4-Tb®, was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, Chicago, IL 60607, USA.
authors.forename: Nivriti
authors.initials: N
authors.lastname: Gahlaut
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Miller
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D005063:Europium / Q000032:analysis; D007089:Image Enhancement; D028581:Lanthanoid Series Elements / Q000032:analysis*; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods*; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D008863:Microspheres; D013997:Time Factors
chemical_list: D028581:Lanthanoid Series Elements; D005063:Europium
references.citation: Ann N Y Acad Sci. 2008;1130:106-16
references.pmid: 18596339
references.citation: Clin Chem. 2001 Mar;47(3):561-8
references.pmid: 11238312
references.citation: Cytometry A. 2007 Oct;71(10):797-808
references.pmid: 17868086
references.citation: J Histochem Cytochem. 1999 Feb;47(2):183-96
references.pmid: 9889254
references.citation: Anal Chem. 2010 Mar 15;82(6):2529-35
references.pmid: 20151708
references.citation: Cytometry. 1994 Jul 1;16(3):187-94
references.pmid: 7924687
references.citation: Biophys J. 1990 Feb;57(2):313-24
references.pmid: 2317553
references.citation: J Photochem Photobiol B. 2000 Sep;57(2-3):83-9
references.pmid: 11154087
references.citation: Biophys J. 1991 Dec;60(6):1374-87
references.pmid: 1723311
references.citation: Acc Chem Res. 2009 Apr 21;42(4):542-52
references.pmid: 19323456
references.citation: Angew Chem Int Ed Engl. 2009;48(27):4990-2
references.pmid: 19492378
references.citation: J Am Chem Soc. 2003 Nov 5;125(44):13324-5
references.pmid: 14583005
references.citation: J Histochem Cytochem. 1997 Sep;45(9):1279-92
references.pmid: 9283615
references.citation: Nat Protoc. 2008;3(11):1809-14
references.pmid: 18974739
references.citation: Cytometry A. 2006 Aug 1;69(8):940-6
references.pmid: 16969811
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16071-6
references.pmid: 18852476
references.citation: J Microsc. 2007 Dec;228(Pt 3):390-405
references.pmid: 18045334
references.citation: Chem Rev. 2010 May 12;110(5):2729-55
references.pmid: 20151630
references.citation: J Am Chem Soc. 2007 Nov 7;129(44):13502-9
references.pmid: 17927176
references.citation: Cytometry. 1992;13(6):561-70
references.pmid: 1451588
references.citation: Biophys J. 1994 Sep;67(3):957-65
references.pmid: 7811952
references.citation: Cytometry A. 2006 Aug 1;69(8):767-78
references.pmid: 16969794
references.citation: Chem Commun (Camb). 2008 Jan 21;(3):365-7
references.pmid: 18399209
references.citation: Methods Enzymol. 1987;149:42-8
references.pmid: 3695968
references.citation: Cell. 1982 May;29(1):33-41
references.pmid: 6179631
references.citation: Anal Biochem. 1991 Jun;195(2):283-9
references.pmid: 1750681
references.citation: Biophys J. 1998 May;74(5):2210-22
references.pmid: 9591648
references.citation: Cytometry A. 2006 Sep 1;69(9):1020-7
references.pmid: 16888769
references.citation: Cytometry. 1992;13(4):329-38
references.pmid: 1326429
references.citation: J Biomed Opt. 2008 Nov-Dec;13(6):064001
references.pmid: 19123648
references.citation: Anal Chem. 2001 Apr 15;73(8):1869-76
references.pmid: 11338604
references.citation: Cytometry. 1990;11(7):784-92
references.pmid: 2272243
references.citation: Acc Chem Res. 2009 Jul 21;42(7):925-37
references.pmid: 19191558
references.citation: Org Biomol Chem. 2008 Nov 21;6(22):4125-33
references.pmid: 18972043
references.citation: Annu Rev Biophys Biomol Struct. 2002;31:275-302
references.pmid: 11988471
references.citation: Clin Chem. 1993 Sep;39(9):1953-9
references.pmid: 8375081
references.citation: Anal Bioanal Chem. 2009 May;394(1):107-20
references.pmid: 19283368
references.citation: Cytometry. 1996 Aug 1;24(4):312-20
references.pmid: 8866215
references.citation: Cytometry A. 2007 Oct;71(10):783-96
references.pmid: 17868087
references.citation: Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10024-8
references.pmid: 7937831
references.citation: Annu Rev Biochem. 1998;67:509-44
references.pmid: 9759496
references.citation: J Microsc. 2002 Jun;206(Pt 3):233-8
references.pmid: 12067368
references.citation: Cytometry. 1994 Jul 1;16(3):195-205
references.pmid: 7924688
references.citation: Cytometry. 1994 Jun 1;16(2):113-7
references.pmid: 7924679
references.citation: J Biomed Opt. 2009 Mar-Apr;14(2):024022
references.pmid: 19405752
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 3;107(31):13582-7
references.pmid: 20643966
references.citation: J Am Chem Soc. 2008 Mar 26;130(12):3714-5
references.pmid: 18321106