Abstract
Laser scanning microscopy (LSM) is a technology that allows for direct observations of host-pathogen interactions during infection. Two of the most available forms of LSM are confocal and two-photon LSM. In addition to high resolution and contrast, these two technologies also provide high excitation penetrance in unsectioned samples. High penetrance allows for imaging of layers of tissue that are difficult to image with other more conventional microscopy approaches. Thus, confocal and two-photon LSM open the possibility of observing infection in a three-dimensional context, where the natural architecture of a tissue is preserved. Few studies have used LSM technology to gain insights into Yersinia pestis pathogenesis in the mammalian host. The use of LSM in the plague field has an enormous potential for the discovery of the mechanisms that lie behind key aspects of pathogenesis such as colonization, dissemination, and tissue damage. This chapter provides guidance for the implementation of confocal or two-photon LSM to study Y. pestis interactions with the host in unsectioned tissues. This document provides specific instructions applied to imaging of Y. pestis, and also discusses relevant aspects of imaging, such as the operation of laser scanning microscopes and the use of fluorescent probes.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fischer RS, Wu Y, Kanchanawong P, Shroff H, Waterman CM (2011) Microscopy in 3D: a biologist’s toolbox. Trends Cell Biol 21:682–691
Gonzalez RJ, Lane MC, Wagner NJ, Weening EH, Miller VL (2015) Dissemination of a highly virulent pathogen: tracking the early events that define infection. PLoS Pathog 11:e1004587
Shannon JG, Bosio CF, Hinnebusch BJ (2015) Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas. PLoS Pathog 11:e1004734
Shannon JG, Hasenkrug AM, Dorward DW, Nair V, Carmody AB, Hinnebusch BJ (2013) Yersinia pestis subverts the dermal neutrophil response in a mouse model of bubonic plague. MBio 4:e00170–e00113
Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940
Miyawaki A, Shcherbakova DM, Verkhusha VV (2012) Red fluorescent proteins: chromophore formation and cellular applications. Curr Opin Struct Biol 22:679–688
Richardson DS, Lichtman JW (2015) Clarifying tissue clearing. Cell 162:246–257
Ariel P (2017) A beginner’s guide to tissue clearing. Int J Biochem Cell Biol 84:35–39
Azaripour A, Lagerweij T, Scharfbillig C, Jadczak AE, Willershausen B, Van Noorden CJF (2016) A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue. Prog Histochem Cytochem 51:9–23
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909
Hadjantonakis A-K, Dickinson ME, Fraser SE, Papaioannou VE (2003) Technicolour transgenics: imaging tools for functional genomics in the mouse. Nat Rev Genet 4:613–625
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Acknowledgments
The author would like to thank Virginia L. Miller for kindly reviewing and editing this document.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gonzalez, R.J. (2019). Laser Scanning Microscopy of Yersinia pestis Infected Tissues. In: Vadyvaloo, V., Lawrenz, M. (eds) Pathogenic Yersinia. Methods in Molecular Biology, vol 2010. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9541-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9541-7_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9540-0
Online ISBN: 978-1-4939-9541-7
eBook Packages: Springer Protocols