Research Highlight
Examples of nervous tissue prepared by a combined chemical fixation and HPF.
(A, B) Hippocampal slices that have been chemically fixed prior to HPF.
(C) Cerebellar slices showing good preservation of synaptic vesicles and PSD and (D) mitochondria with attached smooth and rough endoplasmic reticulum. Note the ribosomes in the background of (D).
(E) Spinal root prepared by combined chemical fixation and HPF. Note the smoothness of the membranes and the level of detail in the paranodal loops and axonal-glial junctions. A 3x magnification is shown in (F).
NCMIR’s improved cryofixation protocols for electron tomography are helping to visualize the 3D proteomics of brain tissue
March 2008 — Visualizing the complex cellular organization and structures that bridge the molecular and cellular scales of the central nervous system is a focus of NCMIR efforts. Leveraging electron tomography's powerful ability to resolve a 3D structures down to within tens of nanometers, however is not without challenges. One challenge comes from the requirement of optimally preserving the ultrastructure of relatively thick sections of cells and tissues for electron tomography. We have been systematically investigating procedures to preserve the fine ultrastructure in relatively large neural tissue samples. High pressure freezing (HPF) has been used to provide superior structural preservation of cells and some smaller tissue samples for electron tomography. Harnessing HPF’s advantage for freezing relatively thick tissues (~200-500µm) without significant ice crystal damage, NCMIR’s researchers have optimized procedures in order to preserve relatively thick neural sections for subsequent serial section tomography with minimal artifacts.
HPF offers rapid freezing of cells and tissues, immobilizing samples immediately in milliseconds, compared with chemical fixation’s seconds-to-minutes timescale. Cryofixation from “live” tissue has been employed widely for the preparation of cultured cells, small microorganisms, and more easily accessible tissues such as skin. Such tissues are highly amenable for resin-embedded tomography using HPF; however, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation can be significant. For this reason, NCMIR researchers continue to explore using chemical fixation followed immediately by HPF and also optimizing freeze substitution methods for preserving and staining neural tissue structures contained within specimens of relatively large volume. Embedding these specimens into plastic allows the use of conventional electron microscopy without the inherent technical challenges or dose-sensitivities associated with EM analysis of vitrified specimens.
By studying the ultrastructure of Flock House Virus-infected insect cells, NCMIR’s scientists have compared conventional fixation with HPF methods. Flock House Virus infected S2 cells are an excellent test specimen with key morphological structures that act as hallmarks of optimal specimen preservation. They’ve found that aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone and very close to that of unfixed tissues (“frozen from life”). Using a variety of nervous system tissues, including neurons injected with a fluorescent dye that was photooxidized, NCMIR researchers have shown excellent brain tissue preservation when compared to chemical fixation alone. The subcellular organization is remarkably well-preserved indicating that chemical fixation is not the limiting step for these tissues. In addition, they extended this technique to selectively filling hippocampal and cerebellar neurons in brain slices with Lucifer Yellow that were then photoconverted and demonstrating that preservation is improved even in samples that are processed for selective staining prior to freezing.
NCMIR’s efforts to harness HPF for large volume specimens have paid off. They are presently improving these methods to localize labeled proteins within macromolecular complexes in their cellular environments and more fully explore their functional interactions, a process that is being called “visual proteomics.” Future improvements to NCMIR protocols will include implementation for ReAsH labeled and photoconverted cells and organelles whereby these probes will allow for selective staining of proteins for correlated light and electron microscopy.
Related Publications
Lanman J, Crum J, Deerinck TJ, Gaietta GM, Schneemann A, Sosinsky GE, Ellisman MH and Johnson JE (2007) Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy. J. Struct. Biol.
Perkins, G.A., Sosinsky, G.E., Ghassemzadeh, S., Jones, Y.Z. and Ellisman, M.H. (2007) Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the Node of Ranvier in peripheral nerves. J. Struct. Biol. Oct 22 [Epub ahead of print].
Sosinsky, G.E., Crum, J., Jones, Y.Z., Lanman, J., Smarr, B., Terada, M., Martone, M.E., Deerinck, T.J., Johnson, J.E., Ellisman, M.H. (2007) The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications. J. Struct. Biol., [Epub ahead of print].
