High-Pressure Freezing / Freeze-Substitution System

Grants and Contracts Details


Instrument requested. We request funds to purchase Leica EM ICE High-Pressure Freezing (HPF) system with Leica AFS2 automatic Freeze-Substitution (FS) system (see the detail quote in the Equipment section). This HPF/FS system has been chosen based on several considerations. First, NIH-funded major users on this application require excellent preservation of ultrastructural features in different, often relatively thick, tissues without artifacts of fixation and dehydration. As of today, it can be achieved only by cryo-fixation with high-pressure freezing. Following high-pressure freezing, the samples may be cryo-sectioned and visualized directly by cryo-EM or freeze-substituted and low-temperature embedded into the resin for subsequent processing at room temperature. After survey of the needs of NIH-funded PIs, we have determined that freeze-substitution with low-temperature embedding serves most of these needs. Although direct cryo-EM observations have a better resolution, the convenience of handling the embedded blocks at room temperature surpass some potential loss of resolution for all major projects (see below). Most of our PIs are familiar with ultramicrotome sectioning and “classical” TEM and, therefore, could handle the embedded samples after freeze-substitution. Moreover, the projects of at least three major users (Gentry, Dutch, and Whiteheart) require immunolabeling. Therefore, we aim for the “gold” standard in EM immunolabeling, which is the labelling of post-embedded thin sections at room temperature. Another essential consideration in choosing high-pressure freezing/freeze-substitution system was the immediate impact of this system on the projects of our NIH-funded PIs. The Electron Microscopy Center at the University of Kentucky has recently acquired the FEI Helios Nanolab 660 dual beam instrument, which combines Focused Ion Beam (FIB) milling with Scanning Electron Microscopy (SEM) and low-voltage Scanning Transmission Electron Microscopy (STEM) imaging. Therefore, serial sections of freeze-substituted low-temperature embedded samples could be easily obtained using already available FIB-SEM instrument. It would provide an immediate benefit to the NIH-funded projects of our major users, since the conventional serial sectioning for TEM is extremely time-consuming. At least three major users (Frolenkov, Rabchevsky, and Whiteheart) already use this workflow routinely in their projects (see below), albeit with some limitations due the lack of high-pressure freezing/freeze-substitution capabilities in the Electron Microscopy Center at the University of Kentucky. The proposed high-pressure freezing/freeze-substitution system will be installed in this Center, in the room that has already a dedicated Nikon binocular microscope for dissection of biological samples, an ultramicrotome for sectioning of the embedded blocks for TEM, the dedicated upright microscope to examine thick sections, as well as the tools for mounting and trimming the embedded blocks for FIB-SEM. FEI Helios 660 FIB-SEM and JEOL 2010F TEM are located in the adjacent rooms. Thus, the proposed equipment would allow seamless workflow from hydrated biological tissues to FIB-SEM or TEM imaging. Having in mind the expertise of our Major Users, we expect a minimal training before this workflow become operational. We have considered an alternative to high-pressure freezing/freeze-substitution workflow, which is the high-pressure freezing followed by cryo-sectioning and direct cryo-EM observations. This workflow is more expensive, because it would require acquisition of a cryo-ultramicrotome and replacing of an existing JEOL 2010F TEM, which has no cryo-EM capabilities. We concluded that high-pressure freezing with subsequent freeze-substitution is optimal in our environment and represents a first cost-effective step toward contemporary EM sample preparation techniques. Our choice of equipment is also forward-thinking. At least three of our NIH-funded PI’s (Frolenkov, Smith, Whiteheart) are interested in future ultrastructural studies that would require electrical or optical stimulation of the tissues/cells before freezing. Although the currently funded projects do not require these capabilities and therefore the proposed system does not include them, these options could be added later to Leica EM ICE with minimal cost and system downtime.
Effective start/end date6/15/186/14/19


  • Office of the Director: $294,376.00


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.