Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement.
We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.
Bouwer
James C
JC
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California at San Diego, La Jolla, CA 92093-0608, USA. jbouwer@ncmir.ucsd.edu
Mackey
Mason R
MR
Lawrence
Albert
A
Deerinck
Tom J
TJ
Jones
Ying Z
YZ
Terada
Masako
M
Martone
Maryann E
ME
Peltier
Steven
S
Ellisman
Mark H
MH
D000818:Animals; D019411:Clinical Laboratory Techniques; D003712:Dendrites / Q000648:ultrastructure; D004583:Electrons; D006624:Hippocampus / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008854:Microscopy, Electron; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation / Q000379:methods*; D015233:Models, Statistical; D008962:Models, Theoretical; D012542:Scattering, Radiation; D013194:Staining and Labeling; D014054:Tomography / Q000379:methods*