provided effective EM labels, although they are not yet useable with samples that
have been fixed by rapid freezing and freeze-substitution. An electron dense label
that was usable in the volume of a well-preserved cell would therefore be a significant advance.
The use of fluorescent, clonable labels, such as GFP, has revolutionized light
microscopy where tagged proteins of interest can be observed in a living cell.
Methods to preserve GFP signal after sample processing for electron microscopy
have been used for correlative light and electron microscopy [37, 38]. The
fluorescent signal present in sections of embedded material is correlated to a
structure in the EM through the use of alignment markers, although the label itself
is not visible. Recently, metallothionein (MTH) has been successfully used as a
clonable tag for protein localization in the EM [39]. Heavy metals that bind to MTH
are quite toxic to living cells but their addition can be accomplished during
freeze-substitution or by soaking sections of material embedded in a hydrophilic
resin, such as Lowicryl K4 M. The method works best with proteins that are present
in high copy number. With this approach, a MTH-tagged component of the yeast
spindle pole body (SPC42) was successfully labeled and localized with impressive
signal-to-noise and resolution. However, the method was not successful with protein components in the yeast nuclear pore complex. Therefore, coupling protein
localization with 3-D structure studies of optimally-preserved cells remains a goal
for future structural cell biologists; the availability of such a method would certainly
enhance our understanding of structure-function relationships in cell biology.
Acknowledgements The authors would like to dedicate this chapter to the life and memory of
Peter van der Heide who was instrumental in the early development of these methods. We would
like to thank Mary Morphew for specimen preparation. This work was supported by and NIH
RR00592 and P41GM103431 to A. Hoenger and by NIH/NIBIB grant R01-EB005027 to D.
N. Mastronarde
References
1. D.N. Mastronarde, Automated electron microscope tomography using robust prediction of
specimen movements. J. Struct. Biol. 152(1), 36–51 (2005)
2. J.R. Kremer, D.N. Mastronarde, J.R. McIntosh, Computer visualization of three-dimensional
image data using IMOD. J. Struct. Biol. 116(1), 71–76 (1995)
3. C.J. Peddie, L.M. Collinson, Exploring the third dimension: volume electron microscopy
comes of age. Micron 61, 9–19 (2014)
4. A. Kremer, S. Lippens, S. Bartunkova, B. Asselbergh, C. Blanpain, M. Fendrych, A.
Goossens, M. Holt, S. Janssens, M. Krols, J.-C. Larsimont, C. Mc Guire, M.K. Nowack, X.
Saelens, A. Schertel, B. Schepens, M. Slezak, V. Timmerman, C. Theunis, R. VAN Brempt,
Y. Visser, C.J. Guérin, Developing 3D SEM in a broad biological context. J. Microsc. 259(2),
80–96 (2015)
5. J.R. McIntosh, K.L. McDonald, M.K. Edwards, B.M. Ross, Three-dimensional structure of
the central mitotic spindle of Diatoma vulgare. J. Cell Biol. 83(2 Pt 1), 428–442 (1979)
114
E. O’Toole et al.
have been fixed by rapid freezing and freeze-substitution. An electron dense label
that was usable in the volume of a well-preserved cell would therefore be a significant advance.
The use of fluorescent, clonable labels, such as GFP, has revolutionized light
microscopy where tagged proteins of interest can be observed in a living cell.
Methods to preserve GFP signal after sample processing for electron microscopy
have been used for correlative light and electron microscopy [37, 38]. The
fluorescent signal present in sections of embedded material is correlated to a
structure in the EM through the use of alignment markers, although the label itself
is not visible. Recently, metallothionein (MTH) has been successfully used as a
clonable tag for protein localization in the EM [39]. Heavy metals that bind to MTH
are quite toxic to living cells but their addition can be accomplished during
freeze-substitution or by soaking sections of material embedded in a hydrophilic
resin, such as Lowicryl K4 M. The method works best with proteins that are present
in high copy number. With this approach, a MTH-tagged component of the yeast
spindle pole body (SPC42) was successfully labeled and localized with impressive
signal-to-noise and resolution. However, the method was not successful with protein components in the yeast nuclear pore complex. Therefore, coupling protein
localization with 3-D structure studies of optimally-preserved cells remains a goal
for future structural cell biologists; the availability of such a method would certainly
enhance our understanding of structure-function relationships in cell biology.
Acknowledgements The authors would like to dedicate this chapter to the life and memory of
Peter van der Heide who was instrumental in the early development of these methods. We would
like to thank Mary Morphew for specimen preparation. This work was supported by and NIH
RR00592 and P41GM103431 to A. Hoenger and by NIH/NIBIB grant R01-EB005027 to D.
N. Mastronarde
References
1. D.N. Mastronarde, Automated electron microscope tomography using robust prediction of
specimen movements. J. Struct. Biol. 152(1), 36–51 (2005)
2. J.R. Kremer, D.N. Mastronarde, J.R. McIntosh, Computer visualization of three-dimensional
image data using IMOD. J. Struct. Biol. 116(1), 71–76 (1995)
3. C.J. Peddie, L.M. Collinson, Exploring the third dimension: volume electron microscopy
comes of age. Micron 61, 9–19 (2014)
4. A. Kremer, S. Lippens, S. Bartunkova, B. Asselbergh, C. Blanpain, M. Fendrych, A.
Goossens, M. Holt, S. Janssens, M. Krols, J.-C. Larsimont, C. Mc Guire, M.K. Nowack, X.
Saelens, A. Schertel, B. Schepens, M. Slezak, V. Timmerman, C. Theunis, R. VAN Brempt,
Y. Visser, C.J. Guérin, Developing 3D SEM in a broad biological context. J. Microsc. 259(2),
80–96 (2015)
5. J.R. McIntosh, K.L. McDonald, M.K. Edwards, B.M. Ross, Three-dimensional structure of
the central mitotic spindle of Diatoma vulgare. J. Cell Biol. 83(2 Pt 1), 428–442 (1979)
114
E. O’Toole et al.