Successful ultra-rapid freezing requires that heat be withdrawn so rapidly from an unfixed, un-cryoprotected sample that no ice crystal damage is visible at the electron microscopic level of analysis. This is not a trivial undertaking, and until recently, the improvements gained through ultrarapid freezing were often paid for in terms of decreased productivity. However, over the last several years, improvements in methodology and the introduction of refined equipment for the ultrarapid freezing of biological samples have significantly improved both the ease of application of the technique and the yield of well preserved samples. The EMS-002 Rapid Immersion Freezer allows for reliable, virtually routine freezing of biological samples.
Features:
- Removable drop anvil assembly
- Specimen holder
- Stainless steel liquid nitrogen Dewar
- Cryogen temperature controller
- Drop release
- Removable cryogen holder for safe disposal of cryogen
- Frozen specimen work station
Process of Ultra-Rapid Sample Cooling
The EMS-002 achieves ultra-rapid cooling by quenching the sample in liquid propane or other suitable cryogen, cooled to near liquid nitrogen temperature, using a stainless steel vacuum insulated Dewar and a copper liquefaction chamber. Liquid nitrogen is poured into the Dewar to a level determined by the user. The initial cooling of the cryogen liquefaction chamber takes about fifteen to twenty minutes.
Gaseous propane is then admitted to the liquefaction chamber slowly enough to allow it to condense and fill the chamber with liquefied propane. The propane is allowed to cool to near liquid nitrogen temperature supervised by the digital temperature controller while a sample is being prepared for freezing. The device requires only about ten minutes to cool to operating temperature after the first filling of propane, and less than five minutes after subsequent fillings.
The operator can increase and decrease the rate of cooling by readjusting the level of liquid nitrogen in the Dewar. Heaters, built into the chamber walls and powered by a temperature controller, evenly warm the propane above its freezing point. The controller's temperature is set for the kind of cryogen being used, -185°C for use with commercial grade propane.
Chemically pure propane (map. 190°C) can also be used, but it is significantly more expensive than commercial grade propane and confers no particular advantage. Ethane and a non-flammable cryogen, SUVA-124 produced by Dupont, can also be used as a cryogen in this instrument. When using flammable cryogens like propane and ethane, we recommend using the EMS-002 in a fume hood.
Sample Preparation
The sample can be prepared using any favorite size EM grid if the system is being used for CryoTEM or Panama hat-shaped specimen carriers for freeze-fracture or freeze substitution. Once the sample is applied to the EM grid for instance, the excess must be carefully blotted away with a small piece of filter paper. The goal is to form a thin meniscus of specimen across the holes in the grid. Once achieved, insert the specimen holder in the “Drop Anvil” of the EMS-002.
For freeze fracture specimens, a thin spacer, typically an EM slot grid, is placed on the top of the sample hat, and a second hat is placed upside down on the sample and serves to prevent the sample from being crushed. If the specimen is large and sturdy enough that it can be supported at its periphery, a hole can be made in one or both hats to allow the propane to directly contact the specimen.
Results and Techniques
The technique provides higher rates of cooling than can be attained by plunging the sample into the cryogen by hand, because of the high velocity and accuracy of the sample entering the cryogen. Approximately 100 meters per second is the optimal plunging speed and provides more efficient heat exchange. The freezing is fast enough to capture rapid events and labile structures that are not seen in chemically fixed material. The technique has been applied to many types of specimens: aqueous suspensions and emulsions, tissue culture cells, suspensions of a wide variety of animal and plant cells and tissues. Specimens frozen with the EMS-002 may be freeze fractured (knife cut or double replica), freeze substituted, frozen thin sectioned or freeze dried. The samples may also be used for Cryo-SEM or TEM.
Temperature Controller
Allows the user to set desired hold temperatures of the cryogen being used. The easy to use controller also has an alarm which can be set to alert the user that the cryogen is warming up and the unit needs attention before the cryogen reaches evaporation temperature.
Recycling
The recycling time of the device is only a few minutes, just long enough to be ready again when processing of the last sample is finished. The device will operate for about two hours on ten litres of liquid nitrogen, including the initial cool down.
Applications:
- Cryofixation of virus partials
- Cryofixation of isolated or assembled macromolecules
- Cryofixation of emulsions, paints and polymers
- Cryofixation of suspensions and tissues for cryo-techniques like freeze fracture/etch, freeze drying, freeze substitution, CryoSEM and CryoTEM
Advantages:
- Economical, universal application rapid freezing unit
- Plunge freezing with temperature controlled cryogen
- Frozen samples handled under cold dry nitrogen atmosphere
- Rapid setup and easy use
- Safe operation and easy cryogen disposal
- Reproducible results
- High and low set points for cryogen control
Specifications:
Dimensions: W279mm x D330mm x H356mm
Electrical: 100-240VAC 50/60Hz 3A
Operational Temperature range: +30 to –200°C
Liquid Nitrogen Consumption: 1-2 litres per hour
Cryogen Volume: 20 mL
Optional Accessories: Ask for price
Climate Controlled Environmental Chamber
Inhibits air-drying and pre-cooling of sample prior to freezing. Allows easy sample blotting to achieve the optimal amount of sample on your grid.
Automatic Liquid Nitrogen Filling
Keeps a constant LN2 level in the Dewar automatically.
Propane Transfer System
Cryo Transfer Face for SEM
Cryo Transfer Face for TEM
Metal Mirror Impact Freezing Fixtures
Allow a quick change from plunge freezing to impact freezing.
Diamond Anvil
Special freeze anvil with real diamond plate for better rapid freezing and easier specimen removal. Does not require polishing like standard copper anvils.
Freeze Forceps
Special self-locking tweezers specifically for use with the plunge freezer. Extra sets allow quicker turn around when freezing multiple samples.
Muscle Biopsy Clamps
Clamps designed for human muscle biopsies to keep the muscle from contracting during fixation. Now it is possible to improve the morphology of the sample through rapid freezing by plunging into liquid cryogen. Fits directly into the drop anvil on the plunge freezer.
The EMS-002 Plunge freezer comes with removable drop anvil assembly, stainless steel liquid nitrogen Dewar, cryogen temperature controller, cryogen holder and instructions. 110V-240V.
Without shaker
Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. K.L. McDonald and R.I. Webb1 have now introduced a new method for freeze substitution with a basic kit that we are proud to offer.
With this unique kit researchers are able to achieve excellent freeze substitution results in as little as 90 minutes, for cells of small volume such as bacteria and tissue culture cells. For those cells of greater volume or that have significant diffusion barriers, such as cuticles or thick cell walls, one can extend the time to 3 hours simply by putting a lid on the box.
Why does it work?
- In well-frozen samples the water molecules are not likely to move around very much, even as the temperatures rise to a point where you would expect hexagonal ice (Dubochet, 2007).
- Agitation should speed up the substitution of acetone for water molecules in much the same way that agitation speeds up development of film.
- Water in the substitution mixture does not appear to slow down substitution as was once believed. In fact, it is known to help membrane contrast (Buser and Walther, 2008).
- K.L. McDonald* and R.I. Webb**, *Electron Microsocpe Laboratory, University of California , Berkley, CA and ** Centre for Microscopy an Microanalysis, University of Queensland, Queensland, Australia
- Other references are listed in the user note.
Kit components
Heater block for 12mm tubes (13mm block is also available)
Ice chest
Cryotubes
Temperature probe
Datalogger
These components can also be ordered separately - ask for price.
For the shaker we recommend EA354-10 orbital platform shaker on page E9
The PELCO® UVC3 Cryo Chamber has been specifically designed to provide an economical yet precisely controlled low temperature environment for polymerization of polymers and resins for cryo specimen preparation. It can also be used for dehydration and infiltration of cryo specimens. This simple and functional unit has a large insulated chamber and comprises fully automatic temperature control for the polymerization process of embedding resins and polymers.
Features
- Liquid crystal display and PLC control
- Fan cooled electronic ballast for UV lamp tubes
- Temperature range can be set from -10°C to -40°C
- Includes memory function for Min/Max temperatures during any run
- 365nm ultraviolet lamps thermally shielded by dual pane Plexiglas
- Holds up to 66 samples with BEEM® Cryo Capsule Holders
- Perfect for Unicryl, LR White, LR Gold, Lowicryls, JB-4, Quetols and all other resins cured by 365nm UV radiation
- Universal voltage
- 3 BEEM® cryo capsule holders included. More on page R2
Operation
- Add dry ice to the chamber
- Set temperature (-10°C to -40°C / 14°F to -40°F)
- Turn on UV lamp and cure embedding resins
Specifications
| UV Light Source | Two 15-watt self-filtering long wavelength tubes 1600 µW/cm_ of 365nm UV radiation at 150mm |
| Temperature Control | -10°C to -40°C Typically ± 1.5 °C |
| Control Type | LCD Display with PLC with function buttons |
| Exterior Dimensions | 641 L x 368 W x 489mm H |
| Interior Dimensions | 565 L x 305 W x 337mm H |
| Power Requirements | 110 - 240VAC, 50/60Hz (240V-0.5A, 110V-1A) |
| Weight | 11.4 kg / Shipping weight: 18.2kg |
Liquid‐proof cryo gloves are specifically designed to maximise safety and comfort for people working in cryogenic environments. They are suitable for handling liquid nitrogen and will protect against splashes, however they are not intended for immersion in liquid nitrogen or other cryogenic liquids.
The outer shell is made of nylon taslan® laminated to a PTFE film. The lining consists of several layers including polyester knit lining for hand, polyolefin hand and cuff insulation and nylon taffeta cuff lining.
Temperature range: ‐160°C to 148°C. Length of gloves: wrist 300mm, mid arm 350‐380mm, elbow 460‐510mm, shoulder 680‐710mm.
Features
- Superior thermal protection
- Comfortable, lightweight, excellent dexterity
- Breathable, stay‐dry lining
- Protection from splashes of cryogenic liquids
- Machine washable
- Steam autoclaveable (4% shrinkage)
Applications
- Ultra low and blast freezers
- Cold rooms
- Cryo freezers
- Dry ice handling
Environments
- Clinical laboratory
- Bio‐medical
- Blood banks
- Frozen food processing
- Pharmaceutical
Flexible, low temperature resisitant, waterproof and breathable.
These liquid‐proof cryo gloves are specifically designed to maximise safety and comfort for people working with cryogenic processes. They are suitable for handling liquid nitrogen and will protect against splashes, however they are not intended for immersion in liquid nitrogen or other cryogenic liquids as these may penetrate perfectly waterproof gloves. These cryo gloves are designed to work at liquid nitrogen temperatures about ‐195°C. The cryo gloves are bulky and a precise fit is not required. Purchase this glove with a larger than required palm widths and with the required lengths.
- Superior thermal protection
- Comfortable, lightweight, excellent dexterity
- Breathable, stay‐dry lining
- Protection from splashes of cryogenic liquids
- Machine washable
- Steam autoclaveable (4% shrinkage)
- Ultra low and blast freezers
- Cold rooms
- Cryo freezers
- Dry ice handling
- Clinical laboratory
- Bio‐medical
- Blood banks
- Frozen food processing
- Pharmaceutical
Wrist: 320mm
Mid arm: 380mm
Elbow: 480mm
Shoulder: 680mm
Width of palm: 140mm
Middle finger length: 90mm
Middle finger width: 32mm