High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues
- University of California, San Diego, United States
Abstract
Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.
Data availability
A source data file has been provided for Figure 4 (Figure 4-source data 1). The SBEM volume of a Drosophila antenna presented in this study has been deposited to the Cell Image Library. The SBEM volume, the tdTomato confocal volume and the DRAQ5 confocal volume used for 3D CLEM in a mouse brain (corresponding to Figure 5) have also been deposited to the Cell Image Library. The video of 3D CLEM in a mouse brain expressing tdTomato that corresponds to Figure 5-video supplement 1 has been deposited to the Cell Image Library.
-
SBEM of Drosophila antenna (10x UAS-myc-APEX2-Orco; Or47b-GAL4)Publicly available at the Cell Image Library under accession number CIL:50452.
-
SBEM volume used for 3D CLEM in mouse brainPublicly available at the Cell Image Library under accession number CIL:50451.
-
tdTomato confocal volume used for 3D CLEM in a mouse brainPublicly available at the Cell Image Library under accession number CIL:50453.
-
DRAQ5 confocal volume used for 3D CLEM in a mouse brainPublicly available at the Cell Image Library under accession number CIL:50454.
-
Video of 3D CLEM in a mouse brain expressing tdTomatoPublicly available at the Cell Image Library under accession number CIL:50401.
Article and author information
Author details
Funding
National Institute on Deafness and Other Communication Disorders (R01DC015519)
- Chih-Ying Su
National Institute of General Medical Sciences (P41GM103412)
- Mark H Ellisman
Croucher Foundation
- Tin Ki Tsang
Kavli Foundation (2015-004)
- Chih-Ying Su
- Mark H Ellisman
Ray Thomas Edwards Foundation
- Chih-Ying Su
Frontiers of Innovation Scholars Program
- Tin Ki Tsang
National Institute of General Medical Sciences (R01GM086197)
- Daniela Boassa
Kavli Foundation (2016-038)
- Daniela Boassa
- Davide Dulcis
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Tsang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 8,542
- views
-
- 1,315
- downloads
-
- 63
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues
eLife 7:e35524.
https://doi.org/10.7554/eLife.35524
Further reading
-
- Neuroscience
Unipolar brush cells (UBCs) are excitatory interneurons in the cerebellar cortex that receive mossy fiber (MF) inputs and excite granule cells. The UBC population responds to brief burst activation of MFs with a continuum of temporal transformations, but it is not known how UBCs transform the diverse range of MF input patterns that occur in vivo. Here, we use cell-attached recordings from UBCs in acute cerebellar slices to examine responses to MF firing patterns that are based on in vivo recordings. We find that MFs evoke a continuum of responses in the UBC population, mediated by three different types of glutamate receptors that each convey a specialized component. AMPARs transmit timing information for single stimuli at up to 5 spikes/s, and for very brief bursts. A combination of mGluR2/3s (inhibitory) and mGluR1s (excitatory) mediates a continuum of delayed, and broadened responses to longer bursts, and to sustained high frequency activation. Variability in the mGluR2/3 component controls the time course of the onset of firing, and variability in the mGluR1 component controls the duration of prolonged firing. We conclude that the combination of glutamate receptor types allows each UBC to simultaneously convey different aspects of MF firing. These findings establish that UBCs are highly flexible circuit elements that provide diverse temporal transformations that are well suited to contribute to specialized processing in different regions of the cerebellar cortex.
-
- Neuroscience
We hypothesized that active outer hair cells drive cochlear fluid circulation. The hypothesis was tested by delivering the neurotoxin, kainic acid, to the intact round window of young gerbil cochleae while monitoring auditory responses in the cochlear nucleus. Sounds presented at a modest level significantly expedited kainic acid delivery. When outer-hair-cell motility was suppressed by salicylate, the facilitation effect was compromised. A low-frequency tone was more effective than broadband noise, especially for drug delivery to apical locations. Computational model simulations provided the physical basis for our observation, which incorporated solute diffusion, fluid advection, fluid–structure interaction, and outer-hair-cell motility. Active outer hair cells deformed the organ of Corti like a peristaltic tube to generate apically streaming flows along the tunnel of Corti and basally streaming flows along the scala tympani. Our measurements and simulations coherently suggest that active outer hair cells in the tail region of cochlear traveling waves drive cochlear fluid circulation.
