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Developing 3D SEM in a broad biological context

Anna Kremer (UGent) , Saskia Lippens (UGent) , Sona Bartunkova (UGent) , Bob Asselbergh (UGent) , C Blanpain, Matyas Fendrych (UGent) , Alain Goossens (UGent) , M Holt, Sophie Janssens (UGent) , Michiel Krols (UGent) , et al.
(2015) JOURNAL OF MICROSCOPY. 259(2). p.80-96
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Ghent researchers on unfolded proteins in inflammatory disease (GROUP-ID)
Abstract
When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions. Lay Description Life happens in three dimensions. For many years, first light, and then EM struggled to image the smallest parts of cells in 3D. With recent advances in technology and corresponding improvements in computing, scientists can now see the 3D world of the cell at the nanoscale. In this paper we present the results of high resolution 3D imaging in a number of diverse cells and tissues from multiple species. 3D reconstructions of cell structures often revealed them to be significantly more complex when compared to extrapolations made from 2D studies. Correlating functional 3D LM studies with 3D EM results opens up the possibility of making new strides in our understanding of how cell structure is connected to cell function.
Keywords
focused ion beam scanning electron microscopy, sample preparation, serial block-face scanning electron microscopy, FOCUSED ION-BEAM, TRANSMISSION ELECTRON-MICROSCOPY, CORRELATIVE LIGHT, SCANNING-ELECTRON, IN-VIVO, SYSTEM, CELLS, ULTRASTRUCTURE, ARABIDOPSIS, SAMPLES, Correlative light and electron microscopy

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Chicago
Kremer, Anna, Saskia Lippens, Sona Bartunkova, Bob Asselbergh, C Blanpain, Matyas Fendrych, Alain Goossens, et al. 2015. “Developing 3D SEM in a Broad Biological Context.” Journal of Microscopy 259 (2): 80–96.
APA
Kremer, A., Lippens, S., Bartunkova, S., Asselbergh, B., Blanpain, C., Fendrych, M., Goossens, A., et al. (2015). Developing 3D SEM in a broad biological context. JOURNAL OF MICROSCOPY, 259(2), 80–96.
Vancouver
1.
Kremer A, Lippens S, Bartunkova S, Asselbergh B, Blanpain C, Fendrych M, et al. Developing 3D SEM in a broad biological context. JOURNAL OF MICROSCOPY. 2015;259(2):80–96.
MLA
Kremer, Anna et al. “Developing 3D SEM in a Broad Biological Context.” JOURNAL OF MICROSCOPY 259.2 (2015): 80–96. Print.
@article{6901255,
  abstract     = {When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions. 
Lay Description Life happens in three dimensions. For many years, first light, and then EM struggled to image the smallest parts of cells in 3D. With recent advances in technology and corresponding improvements in computing, scientists can now see the 3D world of the cell at the nanoscale. In this paper we present the results of high resolution 3D imaging in a number of diverse cells and tissues from multiple species. 3D reconstructions of cell structures often revealed them to be significantly more complex when compared to extrapolations made from 2D studies. Correlating functional 3D LM studies with 3D EM results opens up the possibility of making new strides in our understanding of how cell structure is connected to cell function.},
  author       = {Kremer, Anna and Lippens, Saskia and Bartunkova, Sona and Asselbergh, Bob and Blanpain, C and Fendrych, Matyas and Goossens, Alain and Holt, M and Janssens, Sophie and Krols, Michiel and Larsimont, J-C and Mc Guire, Conor and Nowack, Moritz and Saelens, Xavier and Schertel, A and Schepens, Bert and Slezak, M and Timmerman, V and Theunis, C and Van Brempt, R and Visser, Y and Guerin, Chris},
  issn         = {0022-2720},
  journal      = {JOURNAL OF MICROSCOPY},
  keywords     = {focused ion beam scanning electron microscopy,sample preparation,serial block-face scanning electron microscopy,FOCUSED ION-BEAM,TRANSMISSION ELECTRON-MICROSCOPY,CORRELATIVE LIGHT,SCANNING-ELECTRON,IN-VIVO,SYSTEM,CELLS,ULTRASTRUCTURE,ARABIDOPSIS,SAMPLES,Correlative light and electron microscopy},
  language     = {eng},
  number       = {2},
  pages        = {80--96},
  title        = {Developing 3D SEM in a broad biological context},
  url          = {http://dx.doi.org/10.1111/jmi.12211},
  volume       = {259},
  year         = {2015},
}

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