Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB？ m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org.
References
[1]
Masters BR, So PTC (2004) Antecedents of two-photon excitation laser scanning microscopy. Micros Res Tech 63: 3–11.
[2]
K？nig K (2000) Multiphoton microscopy in life sciences. J Microsc 200: 83–104.
[3]
Reyes-Aldasoro CC, Wilson I, Prise VE, Barber PR, Ameer-Beg SM, et al. (2008) Estimation of apparent tumor vascular permeability from multiphoton fluorescence microscopic images of P22 rat sarcomas in vivo. Microcirculation 15: 65–79.
[4]
Theruvath TP, Zhong Z, Pediaditakis P, Ramshesh VK, Currin RT, et al. (2008) Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition. Hepatology 47: 236–246.
[5]
Zhong Z, Theruvath TP, Currin RT, Waldmeier PC, Lemasters JJ (2007) NIM811, a mitochondrial permeability transition inhibitor, prevents mitochondrial depolarization in small-for-size rat liver grafts. Am J Transplantation 7: 1103–1111.
[6]
Lunt SJ, Gray C, Reyes-Aldasoro CC, Matcher S, Tozer GM (2010) Application of Intravital Microscopy in Studies of Tumour Microcirculation. J Biomed Optics 15: 11113.
[7]
Tozer GM, Akerman S, Cross N, Barber PR, Bjorndahl M, et al. (2008) Blood Vessel Maturation and Response to Vascular-Disrupting Therapy in Single Vascular Endothelial Growth Factor-A Isoform–Producing Tumors. Cancer Res 68: 2301–2311.
[8]
Elks PM, van Eeden FJ, Dixon G, Wang X, Reyes-Aldasoro CC, et al. (2011) Activation of Hif-1alpha delays inflammation resolution by promoting neutrophil persistence in a zebrafish inflammation model. Blood 118: 712–722.
[9]
Halbritter F, Vaidya HJ, Tomlinson SR (2012) GeneProf: analysis of high-throughput sequencing experiments. Nat Methods 9: 7–8.
[10]
Domon B, Aebersold R (2006) Challenges and opportunities in proteomics data analysis. Mol Cell Proteomics 5: 1921–1926.
[11]
Renshaw SA, Loynes CA, Trushell DM, Elworthy S, Ingham PW, et al. (2006) A transgenic zebrafish model of neutrophilic inflammation. Blood 108: 3976–3978.
[12]
Xu N, Lei X, Liu L (2011) Tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy. J Vis Exp.
[13]
Kreisel D, Nava RG, Li W, Zinselmeyer BH, Wang B, et al. (2010) In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proc Natl Acad Sci USA 107: 18073–18078.
[14]
Murphy CT, Moloney G, Hall LJ, Quinlan A, Faivre E, et al. (2010) Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis. Clin Exp Immunol 162: 188–196.
[15]
Yoo SK, Starnes TW, Deng Q, Huttenlocher A (2011) Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature 480: 109–112.
[16]
Henrickson SE, Mempel TR, Mazo IB, Liu B, Artyomov MN, et al. (2008) T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation. Nat Immunology 9: 282–291.
[17]
Textor J, Peixoto A, Henrickson SE, Sinn M, von Andrian UH, et al. (2011) Defining the quantitative limits of intravital two-photon lymphocyte tracking. Proc Natl Acad Sci USA 108: 12401–12406.
[18]
Moreau HD, Lemaitre F, Terriac E, Azar G, Piel M, et al. (2012) Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo. Immunity 37: 351–363.
[19]
Mandl JN, Liou R, Klauschen F, Vrisekoop N, Monteiro JP, et al. (2012) Quantification of lymph node transit times reveals differences in antigen surveillance strategies of naive CD4+ and CD8+ T cells. Proc Natl Acad Sci USA 109: 18036–18041.
Grau V, Mewes AU, Alcaniz M, Kikinis R, Warfield SK (2004) Improved watershed transform for medical image segmentation using prior information. IEEE Trans Med Imaging 23: 447–458.
[22]
Liepe J, Taylor H, Barnes CP, Huvet M, Bugeon L, et al. (2012) Calibrating spatio-temporal models of leukocyte dynamics against in vivo live-imaging data using approximate Bayesian computation. Integrat Biol 4: 335–345.
[23]
Taylor HB, Liepe J, Barthen C, Bugeon L, Huvet M, et al.. (2012) P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish. Immunol Cell Biol.
[24]
Fiole D, Touvrey C, Quesnel-Hellmann A, Douady J, Tournier JN (2012) Shape-based tracking allows functional discrimination of two immune cell subsets expressing the same fluorescent tag in mouse lung explant. PLoS One 7: e39831.
[25]
Collins TJ (2007) ImageJ for microscopy. Biotechniques 43: 25–30.
[26]
Meijering E, Dzyubachyk O, Smal I (2012) Methods for cell and particle tracking. Meth Enzymology 504: 183–200.
[27]
Sbalzarini IF, Koumoutsakos P (2005) Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biology 151: 182–195.
[28]
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, et al. (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9: 676–682.
[29]
de Chaumont F, Dallongeville S, Chenouard N, Herve N, Pop S, et al. (2012) Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9: 690–696.
[30]
Winter M, Wait E, Roysam B, Goderie SK, Ali RA, et al. (2011) Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing. Nat Protocols 6: 1942–1952.
[31]
Otsu N (1979) A Threshold selection method from gray level histograms. IEEE Trans Syst Man Cybern 9: 62–66.
[32]
Ground-truth data cannot do it alone. Nat Methods 8: 885–885.
[33]
Nielsen TA, Nilsson H, Matheson T (2012) A formal mathematical framework for physiological observations, experiments and analyses. J R Soc Interface 9: 1040–1050.
[34]
Hellriegel C, Gratton E (2009) Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle tracking. J R Soc Interface 6: S3–S14.
[35]
Nusslein-Volhard C, Dahm R (2002) Zebrafish: A Practical Approach. New York: Oxford University Press.
Cui J, Acton ST, Lin Z (2006) A Monte Carlo approach to rolling leukocyte tracking in vivo. Med Image Anal 10: 598–610.
[38]
Tam J, Roorda A (2011) Speed quantification and tracking of moving objects in adaptive optics scanning laser ophthalmoscopy. J Biomed Opt 16: 036002.
[39]
Walters KB, Dodd ME, Mathias JR, Gallagher AJ, Bennin DA, et al. (2009) Muscle degeneration and leukocyte infiltration caused by mutation of zebrafish Fad24. Dev Dyn 238: 86–99.
[40]
Sirakov NM, Kojouharov H, Sirakova NN (2010) Tracking neutrophil cells by active contours with coherence and boundary improvement filter. In IEEE Image Analysis & Interpretation (SSIAI), 2010 IEEE Southwest Symposium on, Texas, USA.http://dx.doi.org/10.1109/SSIAI.2010.548？3933 Accessed 23 July 2013.
[41]
Mathias JR, Perrin BJ, Liu TX, Kanki J, Look AT, et al. (2006) Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J Leukoc Biol 80: 1281–1288.
[42]
Reyes-Aldasoro CC, Zhao Y, Coca D, Billings SA, Kadirkamanathan V, et al. Analysis of immune cell function using in vivo cell shape analysis and tracking. In: 4th IAPR Int Conf Patt Recog Bioinformatics 2009 7–9 September; Shef, UK. Available http://ml.dcs.shef.ac.uk/old/prib2009/pa？ges/supplementary/Supplementary_papers/p？rib2009_submission_86.pdf Accessed 23July 2013.
[43]
Reyes-Aldasoro CC, Akerman S, Tozer GM (2008) Measuring the velocity of fluorescently labelled red blood cells with a keyhole tracking algorithm. J Microsc 229: 162–173.
[44]
de Oliveira S, Reyes-Aldasoro CC, Candel S, Renshaw SA, Mulero V, et al. (2013) Cxcl8 (IL-8) Mediates Neutrophil Recruitment and Behavior in the Zebrafish Inflammatory Response. J Immunol 190: 4349–59.
[45]
Beltman JB, Maree AF, de Boer RJ (2009) Analysing immune cell migration. Nat Rev Immunology 9: 789–798.
[46]
Kailath T (1967) The Divergence and Bhattacharyya Distance Measures in Signal Selection. IEEE Trans Commun Tech 15: 52–60.
[47]
Reyes-Aldasoro CC, Bhalerao A (2006) The Bhattacharyya space for feature selection and its application to texture segmentation. Patt Recognit 39: 812–826.
[48]
Coleman GB, Andrews HC (1979) Image Segmentation by Clustering. Proc IEEE 67: 773–785.
[49]
Zhang B (2007) Contributions à la microscopie à fluorescence en imagerie biologique : modélisation de la PSF, restauration d’images et détection super-résolutive. PhD Thesis, Paris: école Nationale Supérieure des Télécommunications.
[50]
Luisier F (2010) The SURE-LET Approach to Image Denoising. PhD Thesis, Lausanne: école Polytechnique Fédérale de Lausanne.
[51]
Pase L, Layton JE, Wittmann C, Ellett F, Nowell CJ, et al. (2012) Neutrophil-delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue wounding in zebrafish. Curr Biology : CB 22: 1818–1824.
Schmidschonbein GW, Shih YY, Chien S (1980) Morphometry of Human-Leukocytes. Blood 56: 866–875.
[54]
Tingbeall HP, Needham D, Hochmuth RM (1993) Volume and Osmotic Properties of Human Neutrophils. Blood 81: 2774–2780.
[55]
Rosengren S, Henson PM, Worthen GS (1994) Migration-Associated Volume Changes in Neutrophils Facilitate the Migratory Process in-Vitro. Am J Physiology-Cell Physiology 267: C1623–C1632.
[56]
Ritter M, Schratzberger P, Rossmann H, Woll E, Seiler K, et al. (1998) Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes. B J Pharmacol 124: 627–638.
[57]
Karlsson T, Glogauer M, Ellen RP, Loitto VM, Magnusson KE, et al. (2011) Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization. J Leukoc Biol 90: 963–973.
[58]
Kadirkamanathan V, Anderson SR, Billings SA, Zhang XL, Holmes GR, et al.. (2012) The Neutrophil’s Eye-View: Inference and Visualisation of the Chemoattractant Field Driving Cell Chemotaxis In Vivo. PLoS One 7 e35182. doi:10.1371/journal.pone.0035182.
