title: A novel method for quantified, superresolved, three-dimensional colocalisation of isotropic, fluorescent particles.
abstract: Colocalisation, the overlap of subcellular structures labelled with different colours, is a key step to characterise cellular phenotypes. We have developed a novel bioimage informatics approach for quantifying colocalisation of round, blob-like structures in two-colour, highly resolved, three-dimensional fluorescence microscopy datasets. First, the algorithm identifies isotropic fluorescent particles, of relative brightness compared to their immediate neighbourhood, in three dimensions and for each colour. The centroids of these spots are then determined, and each object in one location of a colour image is checked for a corresponding object in the other colour image. Three-dimensional distance maps between the centroids of differently coloured spots then display where and how closely they colocalise, while histograms allow to analyse all colocalisation distances. We use the method to reveal sparse colocalisation of different human leukocyte antigen receptors in choriocarcinoma cells. It can also be applied to other isotropic subcellular structures such as vesicles, aggresomes and chloroplasts. The simple, robust and fast approach yields superresolved, object-based colocalisation maps and provides a first indication of protein-protein interactions of fluorescent, isotropic particles.
authors: School of Engineering and Computing Sciences, University of Durham, Durham DH1 3LE, UK. boguslaw.obara@durham.ac.uk
authors: Boguslaw
authors: B
authors: Obara
authors: Asma
authors: A
authors: Jabeen
authors: Nelson
authors: N
authors: Fernandez
authors: Pierre Philippe
authors: PP
authors: Laissue
mesh_terms: D000465:Algorithms; D002822:Choriocarcinoma / Q000378:metabolism / Q000473:pathology; D003116:Color; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis*; D006680:HLA Antigens / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D011946:Receptors, Antigen / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D005456:Fluorescent Dyes; D006680:HLA Antigens; D011946:Receptors, Antigen
references: Nat Methods. 2010 Jun;7(6):418-9
references: 20508634
references: Biophys J. 2002 May;82(5):2775-83
references: 11964263
references: Reprod Biomed Online. 2010 Feb;20(2):223-33
references: 20113960
references: Biophys J. 2003 Dec;85(6):4110-21
references: 14645098
references: Proc Natl Acad Sci U S A. 2005 Mar 1;102(9):3360-5
references: 15718280
references: Biol Reprod. 2007 Aug;77(2):274-9
references: 17442853
references: J Microsc. 2011 Jan;241(1):94-100
references: 21118213
references: Curr Protoc Cytom. 2010 Apr;Chapter 12:Unit 12.19.1-20
references: 20373494
references: J Neurosci. 2004 Apr 21;24(16):4070-81
references: 15102922
references: J Immunol. 2008 Mar 15;180(6):4330-7
references: 18322247
references: J Microsc. 2003 Nov;212(Pt 2):122-31
references: 14629561
references: Nature. 2010 Jul 29;466(7306):647-51
references: 20613725
references: Hum Reprod Update. 1999 May-Jun;5(3):234-48
references: 10438108
references: Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11520-5
references: 9326642
references: J Immunol. 2003 Aug 1;171(3):1376-84
references: 12874228
references: J Cell Sci. 1992 Feb;101 ( Pt 2):415-25
references: 1629253
references: J Microsc. 2000 Dec;200(Pt 3):206-17
references: 11106961
references: Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9461-6
references: 10931959
references: J Microsc. 2010 Jan;237(1):12-22
references: 20055915
references: J Lipid Res. 2006 Jul;47(7):1597-8
references: 16645198
references: Histochem Cell Biol. 2012 Jan;137(1):1-10
references: 22086768
references: Nat Methods. 2012 Jun 28;9(7):676-82
references: 22743772
references: Biophys J. 2004 Jun;86(6):3993-4003
references: 15189895
references: IEEE Trans Image Process. 1998;7(1):27-41
references: 18267377
references: J Cell Sci. 1992 Nov;103 ( Pt 3):857-62
references: 1478975
references: Opt Lett. 1988 Jul 1;13(7):547
references: 19745959
references: Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):161-5
references: 8552596
references: Proc Natl Acad Sci U S A. 2007 Oct 30;104(44):17370-5
references: 17959773
references: J Microsc. 2006 Dec;224(Pt 3):213-32
references: 17210054
references: Acta Histochem Cytochem. 2007 Aug 30;40(4):101-11
references: 17898874
references: Microsc Res Tech. 2004 Jun 1;64(2):103-12
references: 15352081
references: Histochem Cell Biol. 2008 Nov;130(5):833-43
references: 18830616
references: Adv Biochem Eng Biotechnol. 2005;95:201-43
references: 16080270
references: Nat Methods. 2012 Jul;9(7):671-5
references: 22930834
references: Biophys J. 1998 May;74(5):2223-6
references: 9591649