Chapter 6
Conjugate Immunofluorescence—SEM
Array Tomography for Studying
Mammalian Synapses and Axons
Kristina D. Micheva and Kristen D. Phend
Abstract Conjugate immunofluorescence—SEM array tomography enables the
imaging of both the molecular content and the ultrastructure of tissues. The method
is based on physical ultrathin serial sectioning, immunostaining and acquiring
fluorescence and electron microscopy images of resin embedded tissues, followed
by computational volume reconstruction and analysis. Conjugate immunofluorescence—SEM array tomography has been used for the study of brain tissue, and in
particular for the characterization of diverse synapses and axons.
6.1 Introduction/Background
Electron microscopy is a powerful tool to study the ultrastructure of biological
tissues; however, it can provide only limited molecular information.
Immunoelectron microscopy uses antibody labelling to identify the presence of
different molecular species, and secondary antibodies conjugated to colloidal gold
of different sizes [1] are most commonly applied to differentiate between two or,
rarely, three primary antibodies [2]. More recently, other probes of various sizes
and shapes, such as colloidal metal particles with different shapes [3], quantum dots
[4] and nanodiamonds [5], have been explored for increased multiplexing
capabilities.
Multiplexing of antibodies is achieved much more easily with immunofluorescence, due to the availability of detector fluorophores of various wavelengths. One
strategy in particular, the use of multiple rounds of immunofluorescent labelling,
opens the possibility of high-level multiplexing. For example, multi-epitope-ligand
K. D. Micheva (&)
Department of Molecular and Cellular Physiology,
Stanford University School of Medicine, Stanford, CA 94305, USA
e-mail: kmicheva@stanford.edu
K. D. Phend
Department of Cell Biology and Physiology, University of North Carolina,
Chapel Hill, NC 27599, USA
© Springer International Publishing AG 2018
E. Hanssen (ed.), Cellular Imaging, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-68997-5_6
149
Conjugate Immunofluorescence—SEM
Array Tomography for Studying
Mammalian Synapses and Axons
Kristina D. Micheva and Kristen D. Phend
Abstract Conjugate immunofluorescence—SEM array tomography enables the
imaging of both the molecular content and the ultrastructure of tissues. The method
is based on physical ultrathin serial sectioning, immunostaining and acquiring
fluorescence and electron microscopy images of resin embedded tissues, followed
by computational volume reconstruction and analysis. Conjugate immunofluorescence—SEM array tomography has been used for the study of brain tissue, and in
particular for the characterization of diverse synapses and axons.
6.1 Introduction/Background
Electron microscopy is a powerful tool to study the ultrastructure of biological
tissues; however, it can provide only limited molecular information.
Immunoelectron microscopy uses antibody labelling to identify the presence of
different molecular species, and secondary antibodies conjugated to colloidal gold
of different sizes [1] are most commonly applied to differentiate between two or,
rarely, three primary antibodies [2]. More recently, other probes of various sizes
and shapes, such as colloidal metal particles with different shapes [3], quantum dots
[4] and nanodiamonds [5], have been explored for increased multiplexing
capabilities.
Multiplexing of antibodies is achieved much more easily with immunofluorescence, due to the availability of detector fluorophores of various wavelengths. One
strategy in particular, the use of multiple rounds of immunofluorescent labelling,
opens the possibility of high-level multiplexing. For example, multi-epitope-ligand
K. D. Micheva (&)
Department of Molecular and Cellular Physiology,
Stanford University School of Medicine, Stanford, CA 94305, USA
e-mail: kmicheva@stanford.edu
K. D. Phend
Department of Cell Biology and Physiology, University of North Carolina,
Chapel Hill, NC 27599, USA
© Springer International Publishing AG 2018
E. Hanssen (ed.), Cellular Imaging, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-68997-5_6
149