Abstract
Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In this chapter, we describe the practical aspects of imaging neutrophils in zebrafish embryos using spinning disk confocal microscopy. Similar setups can also be applied to image other motile cell types and signaling processes in translucent animals or tissues.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Meeker ND, Trede NS (2008) Immunology and zebrafish: spawning new models of human disease. Dev Comp Immunol 32:745–757
Deng Q, Huttenlocher A (2012) Leukocyte migration from a fish eye’s view. J Cell Sci 125:3949–3956
Graf R, Rietdorf J, Zimmermann T (2005) Live cell spinning disk microscopy. Adv Biochem Eng Biotechnol 95:57–75
Wang E, Babbey CM, Dunn KW (2005) Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems. J Microsc 218:148–159
Fischer RS, Wu Y, Kanchanawong P, Shroff H, Waterman CM (2011) Microscopy in 3D: a biologist’s toolbox. Trends Cell Biol 21: 682–691
Salmon WC, Waters JC (2011) CCD cameras for fluorescence imaging of living cells. Cold Spring Harb Protoc 2011:790–802
Murray JM, Appleton PL, Swedlow JR, Waters JC (2007) Evaluating performance in three-dimensional fluorescence microscopy. J Microsc 228:390–405
Westerfield M (2000) The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio), 4th edn. University of Oregon Press, Eugene
Mathias JR, Perrin BJ, Liu TX, Kanki J, Look AT, Huttenlocher A (2006) Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J Leukoc Biol 80:1281–1288
Renshaw SA, Loynes CA, Trushell DM, Elworthy S, Ingham PW, Whyte MK (2006) A transgenic zebrafish model of neutrophilic inflammation. Blood 108: 3976–3978
Hall C, Flores MV, Storm T, Crosier K, Crosier P (2007) The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol 7:42
Lam PY, Yoo SK, Green JM, Huttenlocher A (2012) The SH2-domain-containing inositol 5-phosphatase (SHIP) limits the motility of neutrophils and their recruitment to wounds in zebrafish. J Cell Sci 125:4973–4978
Urasaki A, Morvan G, Kawakami K (2006) Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174: 639–649
Rosen JN, Sweeney MF, Mably JD (2009) Microinjection of zebrafish embryos to analyze gene function. J Vis Exp 25:1115
Yoo SK, Huttenlocher A (2011) Spatiotemporal photolabeling of neutrophil trafficking during inflammation in live zebrafish. J Leukoc Biol 89:661–667
d’Alencon CA, Pena OA, Wittmann C, Gallardo VE, Jones RA, Loosli F, Liebel U, Grabher C, Allende ML (2010) A high-throughput chemically induced inflammation assay in zebrafish. BMC Biol 8:151
Tobin DM, Vary JC Jr, Ray JP, Walsh GS, Dunstan SJ, Bang ND, Hagge DA, Khadge S, King MC, Hawn TR, Moens CB, Ramakrishnan L (2010) The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell 140:717–730
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
Groom D (2002) Cosmic rays and other nonsense in astronomical CCD imagers. Exp Astron 14:45–55
Zhang Y, Bai XT, Zhu KY, Jin Y, Deng M, Le HY, Fu YF, Chen Y, Zhu J, Look AT, Kanki J, Chen Z, Chen SJ, Liu TX (2008) In vivo interstitial migration of primitive macrophages mediated by JNK-matrix metalloproteinase 13 signaling in response to acute injury. J Immunol 181:2155–2164
Acknowledgements
Dr. Benjamin Ng (Carl Zeiss) has provided valuable advice on the SDCM setup. This work was supported by the National Institutes of Health (grant number GM074827) to A. H. and the Hong Kong Croucher Foundation, Joint Universities Summer Teaching Laboratory (JUSTL) program to P-Y.L.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lam, Py., Fischer, R.S., Shin, W.D., Waterman, C.M., Huttenlocher, A. (2014). Spinning Disk Confocal Imaging of Neutrophil Migration in Zebrafish. In: Quinn, M., DeLeo, F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology, vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-845-4_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-844-7
Online ISBN: 978-1-62703-845-4
eBook Packages: Springer Protocols