Abstract
Fluorescence imaging has been applied to various areas of biological research, including studies of physiological, neurological, oncological, cell biological, molecular, developmental, immunological, and infectious processes. In this chapter, we describe methods of fluorescent imaging applied to examination of subcutaneous and pulmonary mycobacterial infections in an animal model. Since slow growth of Mycobacterium tuberculosis (Mtb) hinders development of new diagnostics, therapeutics, and vaccines for tuberculosis (TB), we developed fluorescent protein (FP) expressing mycobacterial strains for in vivo imaging, which can be used to track bacterial location and to quantitate bacterial load directly in living animals. After comparison of imaging data using strains expressing different fluorescent proteins, we found that strains expressing L5-tdTomato display the greatest fluorescence. Here, we describe detailed protocols for tdTomato-labeled M. bovis BCG imaging in real time for subcutaneous and pulmonary infections in living mice. These procedures allow rapid and accurate determination of bacterial numbers in live mice.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
WHO (2015) Global tuberculosis control 2014. WHO, Geneva
Glickman MS, Jacobs WR Jr (2001) Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline. Cell 104(4):477–485
Smith I (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 16(3):463–496
Zumla A, Raviglione M, Hafner R, von Reyn CF (2013) Tuberculosis. N Engl J Med 368(8):745–755. https://doi.org/10.1056/NEJMra1200894
Passamaneck YJ, Di Gregorio A, Papaioannou VE, Hadjantonakis AK (2006) Live imaging of fluorescent proteins in chordate embryos: from ascidians to mice. Microsc Res Tech 69(3):160–167. https://doi.org/10.1002/jemt.20284
Wacker SA, Oswald F, Wiedenmann J, Knochel W (2007) A green to red photoconvertible protein as an analyzing tool for early vertebrate development. Dev Dynam 236(2):473–480. https://doi.org/10.1002/dvdy.20955
Hoffman RM (2005) Advantages of multi-color fluorescent proteins for whole-body and in vivo cellular imaging. J Biomed Opt 10(4):41202. https://doi.org/10.1117/1.1992485
Stewart CN Jr (2006) Go with the glow: fluorescent proteins to light transgenic organisms. Trends Biotechnol 24(4):155–162. doi:S0167-7799(06)00030-8 [pii]10.1016/j.tibtech.2006.02.002
Seitz G, Warmann SW, Fuchs J, Mau-Holzmann UA, Ruck P, Heitmann H, Hoffman RM, Mahrt J, Muller GA, Wessels JT (2006) Visualization of xenotransplanted human rhabdomyosarcoma after transfection with red fluorescent protein. J Pediatr Surg 41(8):1369–1376. https://doi.org/10.1016/j.jpedsurg.2006.04.039
Winnard PT Jr, Kluth JB, Raman V (2006) Noninvasive optical tracking of red fluorescent protein-expressing cancer cells in a model of metastatic breast cancer. Neoplasia 8(10):796–806
Weissleder R (2001) A clearer vision for in vivo imaging. Nat Biotechnol 19(4):316–317
Muller-Taubenberger A, Anderson KI (2007) Recent advances using green and red fluorescent protein variants. Appl Microbiol Biotechnol 77(1):1–12. https://doi.org/10.1007/s00253-007-1131-5
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909
Kong Y, Yang D, Cirillo SL, Li S, Akin A, Francis KP, Maloney T, Cirillo JD (2016) Application of fluorescent protein expressing strains to evaluation of anti-tuberculosis therapeutic efficacy in vitro and in vivo. PLoS One 11(3):e0149972. https://doi.org/10.1371/journal.pone.0149972
Xu H, Rice BW (2009) In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique. J Biomed Opt 14(6):064011. https://doi.org/10.1117/1.3258838
Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, Solovieva EA, Lukyanov KA, Bogdanova EA, Zaraisky AG, Lukyanov S, Chudakov DM (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4(9):741–746
Wang L, Jackson WC, Steinbach PA, Tsien RY (2004) Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci U S A 101(48):16745–16749. https://doi.org/10.1073/pnas.0407752101
Kredel S, Oswald F, Nienhaus K, Deuschle K, Rocker C, Wolff M, Heilker R, Nienhaus GU, Wiedenmann J (2009) mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures. PLoS One 4(2):e4391. https://doi.org/10.1371/journal.pone.0004391
Lin MZ, McKeown MR, Ng HL, Aguilera TA, Shaner NC, Campbell RE, Adams SR, Gross LA, Ma W, Alber T, Tsien RY (2009) Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem Biol 16(11):1169–1179. https://doi.org/10.1016/j.chembiol.2009.10.009
Chu J, Haynes RD, Corbel SY, Li P, Gonzalez-Gonzalez E, Burg JS, Ataie NJ, Lam AJ, Cranfill PJ, Baird MA, Davidson MW, Ng HL, Garcia KC, Contag CH, Shen K, Blau HM, Lin MZ (2014) Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat Methods 11(5):572–578. https://doi.org/10.1038/nmeth.2888
Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, Ward TH, Bancroft GJ, Schaible UE, Parish T (2010) Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. PLoS One 5(3):e9823. https://doi.org/10.1371/journal.pone.0009823
Zelmer A, Carroll P, Andreu N, Hagens K, Mahlo J, Redinger N, Robertson BD, Wiles S, Ward TH, Parish T, Ripoll J, Bancroft GJ, Schaible UE (2012) A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging. J Antimicrob Chemother 67(8):1948–1960. https://doi.org/10.1093/jac/dks161
Kong Y, Akin AR, Francis KP, Zhang N, Troy TL, Xie H, Rao J, Cirillo SLG, Cirillo JD (2011) Whole-body imaging of infection using fluorescence. Curr Protoc Microbiol Chapter 2:Unit 2C.3. https://doi.org/10.1002/9780471729259.mc02c03s21
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kong, Y., Cirillo, J.D. (2018). Fluorescence Imaging of Mycobacterial Infection in Live Mice Using Fluorescent Protein-Expressing Strains. In: Dubey, P. (eds) Reporter Gene Imaging. Methods in Molecular Biology, vol 1790. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7860-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7860-1_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7858-8
Online ISBN: 978-1-4939-7860-1
eBook Packages: Springer Protocols