ABSTRACT
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.
Subject(s)
Clinical Laboratory Techniques , Microscopy, Electron, Transmission/methods , Tomography/methods , Animals , Dendrites/ultrastructure , Electrons , Hippocampus/ultrastructure , Image Processing, Computer-Assisted , Mice , Microscopy, Electron , Microscopy, Electron, Transmission/instrumentation , Models, Statistical , Models, Theoretical , Scattering, Radiation , Staining and LabelingABSTRACT
Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.
