title: Standard fluorescent imaging of live cells is highly genotoxic.
abstract: Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: (a) violet light [340-380 nm], used to excite DAPI and other blue fluorophores, (b) blue light [460-500 nm] commonly used to image green fluorescent protein (GFP) and Calcein AM, and (c) green light [528-553 nm], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant damage levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced GFP or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore, the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time, and fluorophore as parameters that can be modulated to reduce light-induced DNA damage.
authors: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors: Jing
authors: J
authors: Ge
authors: David K
authors: DK
authors: Wood
authors: David M
authors: DM
authors: Weingeist
authors: Somsak
authors: S
authors: Prasongtanakij
authors: Panida
authors: P
authors: Navasumrit
authors: Mathuros
authors: M
authors: Ruchirawat
authors: Bevin P
authors: BP
authors: Engelward
mesh_terms: D002470:Cell Survival / Q000528:radiation effects; D020552:Comet Assay; D004249:DNA Damage; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D019044:Image Cytometry; D007211:Indoles; D008027:Light; D008214:Lymphocytes / Q000166:cytology / Q000528:radiation effects*; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D059010:Single-Cell Analysis
chemical_list: D029968:Escherichia coli Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007211:Indoles; D049452:Green Fluorescent Proteins; C085925:calcein AM; C007293:DAPI; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli
references: J Biol Chem. 2001 Sep 21;276(38):35714-22
references: 11473117
references: J Virol. 2011 Sep;85(18):9268-75
references: 21752918
references: Chem Res Toxicol. 2000 Jul;13(7):541-9
references: 10898585
references: Bioresour Technol. 2011 Apr;102(8):5259-64
references: 21376581
references: Carcinogenesis. 1997 Apr;18(4):811-6
references: 9111219
references: Carcinogenesis. 2000 Mar;21(3):397-404
references: 10688860
references: Mutat Res. 1993 May;299(3-4):135-45
references: 7683082
references: Proc Natl Acad Sci U S A. 1988 Mar;85(5):1586-9
references: 3278320
references: Mol Biotechnol. 2004 Mar;26(3):249-61
references: 15004294
references: Acc Chem Res. 2008 Aug;41(8):1075-83
references: 18666785
references: Biochem Biophys Res Commun. 1984 Aug 30;123(1):291-8
references: 6477583
references: Cytometry A. 2007 Aug;71(8):592-8
references: 17421025
references: Biomed Pharmacother. 2002 Jun;56(4):186-93
references: 12109811
references: J Photochem Photobiol B. 1990 Mar;4(4):349-61
references: 2111381
references: Genetics. 1988 Apr;118(4):551-60
references: 2966755
references: Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):10008-13
references: 20534572
references: DNA Repair (Amst). 2004 Oct 5;3(10):1323-34
references: 15336627
references: Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10124-8
references: 1946433
references: Fertil Steril. 2001 Mar;75(3):588-93
references: 11239546
references: Cancer Res. 2005 Nov 15;65(22):10159-63
references: 16287999
references: Carcinogenesis. 1997 Nov;18(11):2225-31
references: 9395225
references: Photochem Photobiol Sci. 2010 Dec;9(12):1634-9
references: 20931137
references: Acta Biochim Pol. 2001;48(3):611-4
references: 11833769
references: Cytometry A. 2010 Apr;77(4):399-405
references: 20131407
references: Radiat Prot Dosimetry. 2006;122(1-4):124-7
references: 17351270
references: Hum Immunol. 1993 Aug;37(4):264-70
references: 8300411
references: Radiat Res. 2000 Sep;154(3):342-6
references: 11012342
references: Biochem Biophys Res Commun. 1994 Jun 30;201(3):1132-9
references: 8024554
references: Mutat Res. 1991 Jan;246(1):187-91
references: 1986263
references: Biochemistry. 1990 Jul 31;29(30):7024-32
references: 2223758
references: Respir Physiol Neurobiol. 2011 May 31;176(3):110-7
references: 21315850
references: J Exp Med. 1956 May 1;103(5):653-66
references: 13319584
references: J Immunol Methods. 1999 Jun 24;226(1-2):29-41
references: 10410969
references: Curr Protoc Cell Biol. 2010 Dec;Chapter 6:Unit 6.11.
references: 21154553
references: Cytometry. 1995 Oct 1;21(2):160-9
references: 8582236
references: Photochem Photobiol. 1993 Jan;57(1):163-74
references: 8389052
references: Histochem Cell Biol. 2004 Nov;122(5):499-505
references: 15503120
references: Mutagenesis. 1996 Mar;11(2):169-75
references: 8671734
references: Curr Protoc Cell Biol. 2011 Mar;Chapter 26:Unit 26.9
references: 21400700
references: Radiat Res. 2008 Sep;170(3):335-44
references: 18763857
references: Exp Cell Res. 1988 Mar;175(1):184-91
references: 3345800
references: J Biol Chem. 2001 May 25;276(21):17949-57
references: 11278609
references: Mutat Res. 2005 Apr 1;571(1-2):19-31
references: 15748635
references: Nat Protoc. 2006;1(1):23-9
references: 17406208
references: Cancer Res. 1992 Nov 15;52(22):6400-3
references: 1423288
references: Nature. 1991 Jan 31;349(6308):431-4
references: 1992344
references: Radiat Res. 2004 Oct;162(4):405-15
references: 15447042
references: J Photochem Photobiol B. 1989 Oct;4(1):21-34
references: 2509659
references: J Immunol Methods. 1991 Nov 5;144(1):101-15
references: 1960398
references: J Pathol. 2009 Sep;219(1):77-86
references: 19479711
references: FASEB J. 2011 Sep;25(9):3079-91
references: 21613571
references: Am J Physiol Cell Physiol. 2000 Jul;279(1):C147-57
references: 10898726
references: Photochem Photobiol. 1990 Dec;52(6):1119-36
references: 2087500
references: Cytometry A. 2005 Sep;67(1):45-52
references: 16082711
references: Cytometry. 1999 Aug 1;36(4):312-8
references: 10404146
references: Anal Biochem. 2000 Apr 10;280(1):11-9
references: 10805515