Signedon vacuum unit, below: PFEIFFER
on vacuum unit, top: BALZERS
on computer monitor: HITACHI DENSHI LTD.
on CPU: Zilog
on DRAM: Mostek
on synchonous stepping motor: SUPERIOR ELECTRIC
on motor: BODINE / ELECTRIC / COMPANY
Inscribedon front left corner: Harvard University, Biolabs Inventory No.: 021151
FunctionUsed to visualize the ultrastructure of cell compartments and the interior surface of the cell membrane. The freeze-fracturing and etching technique provided a method to examine the ultrastructure of the cell components without altering them chemically (fixation) or physically (dehydration, impregnation with resin, drying). The method involves fracturing a frozen biological material and replicating the contour of the exposed surface with carbon and platinum.
The freeze-fracture & etching technique involves five processes: freezing, fracturing, etching, replica formation, and replica cleaning. A biological specimen to be examined is placed on a copper specimen support and is rapidly frozen by being plunged into liquid Freon, liquefied by liquid nitrogen. The standard specimen support is a "hat shaped" disk, 3 mm in diameter, punched out of a piece of 3 mil (0.076 mm) thick copper. Freon-22 has physical properties (such as low freezing point, high thermal conductivity, high heat capacity, and high boiling point) that maximize the freezing rate to prevent big ice crystals from forming and causing structural disruption in the specimen. The freezing process takes 3-5 seconds. To keep the specimen frozen until it is transferred to the automatic freeze-etching machine, it is stored in a small, perforated Kodak canister that is placed in the Dewar flask containing liquid nitrogen. Once transferred into the vacuum chamber of the machine, the pressure within the chamber is decreased and the specimen is cut with a cold razor blade bolted onto the microtome arm. The knife breaks the specimen along the plane of weakest bonds and exposes the hydrophobic interior of the cell membrane. With the last pass of the knife through the sample, the temperature and the pressure inside the chamber are adjusted to begin the etching process which sublimes volatile components from the fractured specimen surface and exposes additional cell ultrastructure. Finally, the replica of the exposed surface is made by depositing a mixture of platinum and carbon at an angle to the surface by heating (or 'firing') a Pt-C electrode at low voltage and high current. This process is often called 'shadowing.' A higher structural resolution is achieved when the stage is rotated and Pt-C is deposited from multiple angles. The three processes carried out in the machine, fracturing, etching, and replica formation, are automated in Elgbrant (see Biofreeze Automatic Freeze Etching System Manual in Box 2, 1999-1-0041u). The resulting replica is taken out of the machine and floated off onto cleaning solutions, most often bleach and distilled water, to rid the organic materials attached to the replica. A platinum loop is used to transfer the replica from one solution to the next. The washed replica is then placed on an electron microscope grid to be examined under the electron microscope (see figures 1-7 for typical electron microscope images, 1999-1-0041m through s).
Historical AttributesThe "Elgbrant" (for "Elg"saeter and "Brant"on) was designed/created by Professor Daniel Branton, Higgins Professor of Biology at Harvard University, and Dr. Arnljot Elgsaeter, Biophysics faculty member at Norwegian University of Science and Technology in Trondheim, Norway (Dr. Elgsaeter had received his Ph.D. with Prof. Branton at University of California, Berkely in 1971). The designing began in 1978: the electronics were designed and put together in Norway and the frame of the instrument was designed and welded together in Cambridge, MA. In 1980, the electronic parts were brought to Cambridge from Norway and assembled with the frame at the Harvard machine shop by both Dr. Branton and Dr. Elgsaeter. Commercially manufactured products were used for the pumping unit and some of the computer components: Balzers's Pumping unit; Mostek Operating System, Zilog's CPU, Hitachi's monitor, Prowriter's printer. The total cost (including time and cost of raw materials) in making this instrument was about $700,000 in 1970's currency. The Elgbrant became operational in 1986.
Primary SourcesSee the reference list in 1999-1-0041x.
ProvenanceFrom Biolabs Room A-206, Department of Molecular and Cellular Biology, Harvard University. Givent to CHSI by Prof. Daniel Branton.