A gentle, bounce-free assembly for quick-freezing tissues for electron microscopy: Application to isolated torpedine ray electrocyte stacks

An excursion through the ultrastructural world of quick‐frozen pancreatic islets

“…These early attempts met with highly variable results. Today, problems involved in contact-freezing, like shearing effects, tissue crushing, excessive ice crystal formation, and bouncing, have been alleviated by mechanical devices using ultrapure copper at liquid nitrogen (Boyne, 1979) or liquid helium (Heuser et al, 1979) temperature and sapphire at liquid helium temperature (personal communication, Dr. David Coulter, Department of Anatomy, University of Minnesota).…”

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“…We have used the Heuser design because of the high cooling rates obtainable, and the Med-Vac version (instead of the Polaron) due to the improved design of the roller balls (critical for reliable time synchronization), an improved shutter mechanism (effectively preventing precooling) and an improved electromagnet design to avoid millisecond bouncing. An oscilloscope analysis (not shown), similar to the one camed out by Boyne (1979), confirmed that the Med-Vac Cryopress machine is almost free of millisecond bounce.…”

“…Compression can also be a problem. Boyne (1979) found that in electrocyte stacks, the first layer of nerve terminals was always badly damaged, suggesting that such tissues need more specific protection against impact momentum. The equipment developed to minimize these problems is commercially available.…”

“…sample squashing and millisecond bouncing). A recently identified problem (Boyne, 1979) is that the tissue delivery system tends to bounce off the freezing surface faster than the freezing occurs. As the partly cooled tissue loses contact with the block, the cooling rate decreases, with increased ice crystallization; further bounces make the situation worse.…”

“…Bounce is minimized by an electromagnet which catches the freezing head and a compression spring which maintains pressure of the specimen against the cold block during freezing. The other design, the 'Gentleman Jim' (Quick Freezing Devices, Inc.) uses a hydraulic-pneumatic technique for maintaining constant contact of the specimen with the block (Boyne, 1979): a foam-cushioned sample falls towards the copper block which extends into a Dewar full of liquid nitrogen. We have used the Heuser design because of the high cooling rates obtainable, and the Med-Vac version (instead of the Polaron) due to the improved design of the roller balls (critical for reliable time synchronization), an improved shutter mechanism (effectively preventing precooling) and an improved electromagnet design to avoid millisecond bouncing.…”