Biology:Freeze-fracture

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Short description: Freeze-fracture processes and methods

Freeze-fracture is a natural occurrence leading to processes like erosion of the earths crust or simply deterioration of food via freeze-thaw cycles.[1][2] To investigate the process further freeze-fracture is artificially induced to view in detail the properties of materials. Fracture during freezing is often the result of crystallizing water which results in expansion. Crystallization is also a factor leading to chemical changes of a substance due to changes in the crystal surroundings called eutectic formation.[3]

Natural freeze-fracture. An iceberg fractured off a glacier. Erosion reveals the layering hidden within the original glacier

Imaging the fractured surface of a frozen substance allows the interior of the structure to be investigated as illustrated by the picture of a fractured piece of glacier called an iceberg. By photographing at high magnifications more can be learnt about the fractured object's substructure and the changes in the object that occur during freezing. When imaging fractured surfaces in detail, changes occurring during and immediately after fracture as well as sample preparation, must be taken into account if trying to infer the unbroken material's structure.[4][5] The often relatively cold temperatures needed to make an object solid enough to fracture, and the fracture process itself, stress and deform the material. Imaging of fine detail under sub-zero conditions is difficult. The material will start to warm again when removed to a position for photography. Ambient gases, often water vapor, will condense on the cold surfaces, reacting with them, obscuring detail and further warming the object allowing it to reshape.[6][7]

Freezing considerations

Snowflake macro photography

Freezing of a substance is a relative term, often relative to ambient temperatures. Freezing something from liquid or gas phase to a solid allows fracture but has different effects depending on the material involved and how quickly it is frozen. Freezing things slowly allows the material time to re-arrange itself internally. In the example of water, ice forming slowly results in larger crystals leading to a clear glass like substance. If frozen quickly as with snow, the crystals are smaller and less organized, scattering light and appearing white.

Elastic materials

Elastic solids generally become less elastic the cooler they get, making fracturing easier. For example, plastic hoses are flexible on hot days and less flexible and prone to cracking on cold days. Storage of critical items such as blood products in plastic bags must take into account the effect of freezing on the blood but also the changing plasticity of the storage bags.[8] While many synthetic and natural polymers become progressively less elastic with reducing temperature they do not usually crystallize, unless they also contain a free liquid such as water in plants and soils or plasticizers in plastics.

Liquids

Liquids reduced in temperature will become solid enough to be fractured. The abundance of water on earth and particularly in living organisms and soils means frozen water often provides the rigidity needed for and otherwise less brittle object to fracture. While the water increases the rigidity of an object to allow it to be fractured, the formation of ice crystals within an object can also cause significant damage to structures which were previously intact. Changes in the eutectic around the forming crystals is also significant which can be disadvantageous, or used in the case of cooling solder advantageous.[3] Freeze-fracture can occur as part of the freezing process, particularly with liquids that expand as they crystallize such as water. Such fracture is termed pre-fracture.[5]

Two forms of amorphous ice. High density (HDA) top and low density amorphous (LDA) ice bottom

To reduce damage from crystallization cryopreservatives which reduce ice crystal damage are often used but may themselves be toxic to living cells in the concentrations required.[9] For small objects, freezing of liquids can be rapid enough for limited or no crystallization. In the case of water, very rapid freezing leads to vitreous amorphous ice rather than crystalline ice resulting in no detectable damage.[10]

Solids

Perhaps counterintuitively, solids also change into different states when frozen. You may say they become more solid. For example, iron in its various forms will become more brittle at lower temperatures. Steels exhibit low temperature brittleness with a transition temperature from ductile to brittle fracture (TTDB) that varies from about −100 °C to about +100 °C depending on the alloy composition and processing. As the solids change with temperature so does the way they fracture.[11][12]

Gases

Iceplanet

Gases cooled sufficiently will become solid enough to fracture as well, such as with solid carbon dioxide also known as dry ice. Since gases have little structure when used under normal conditions there are currently no investigations into their solid phase structures using freeze-fracture. The requirement for freeze-fracture studies may increase with extra-planetary objects having surface temperatures cold enough for elements that are gases on earth to be naturally solid. Currently only the unfractured structures are being investigated such as solid carbon dioxide on the moon or solid methane and nitrogen on pluto.[13][14][15]

Fracturing considerations

The energy of the fracture

Main page: Physics:Stress (mechanics)

To split a material into two pieces requires the material to be put under enough stress to break it. The amount of stress applied to an object prior to its fracture will determine the amount of energy available for the fracture to take place. Excessive stress results in multiple almost simultaneous fractures, as when shattering a sheet of glass with a hammer. Sufficient but not too much stress normally results in a single fracture. Even with a single fracture any slight excess in stress will lead to fracture that propagates more quickly with more energy and higher temperatures at the fracture face. The higher energy can also result distortions called plastic deformation or even in minute secondary fractures that break fragments off the main fracture face. If the stress is less focused a larger volume will be stressed leading to a slower propagation of the fracture with lower temperatures at the fracture face. Force in excess of that required for a single fracture plane to form is usually released as a combination of significant heating, plastic deformation and secondary fracture.[16]

Temperature

Freeze-fracture and replicated yeast cell at -50°C with two prominent circular bud scars. The ice surrounding the cell melted due to the heat of the fracture, flowed a few microns, and quickly refroze again.

Once cooled sufficiently to fracture, a sample is often cooled further. Stressing and fracturing a sample produces considerable heat, easily enough to thaw a sample again if the temperature is not well below the melting point.[17]

Applications of Freeze-fracture

Here are a few visible examples of freeze-fracture being used directly in our daily lives. There are also less known applications of freeze-fracture knowledge. Better know examples relate to preventing freeze-fracture damage to water supply pipes or engine cooling systems in colder climates.

Daily lives

Ice for cooling

A very common requirement in many peoples' lives is using ice to keep things cool. Large blocks of ice were once the most common way of transporting ice.

Ice blocks are cut and loaded onto a horse drawn sled circa 1935

Once transported the ice would be later fractured into smaller pieces to make it practical to use. Today ice machines produced ready to use ice or ice cubes fractured by blending them into drinks such as a slushy or foods that contain fractured ice such as Ais kacang.

Ais kacang

Tempered glass

Tempered glass has its exterior surface rapidly cooled from the liquid state so that it is frozen solid while the center of the glass is still liquid. As a result, the glass becomes highly stressed. When a single fracture is initiated the considerable stored energy in the prestressing is released. The sudden release of the energy fractures the entire pane into small less damaging pieces, as with a car windscreen.

Engineering

For practical purposes most devices manufactured have an "operating temperature". Often these relate to fragility induced by lowering temperatures increasing the likely-hood of prefracture or fracture.

Steel

Fracture failure of a steel pipe due to cold temperatures

Iron and its various alloys including steel, undergo changes in their resistance to fracturing with temperature. These changes can occur at higher temperatures as the steel solidifies during manufacture and also again at lower temperatures below 100 °C including below the freezing point of water at 0 °C. This has implications in the design and building of steel civil engineering structures such as bridges, buildings and pipes.[11]

Composite materials

Concrete fracture

Freeze-thaw cycles of composite materials can weaken them.[18] Moisture within composite materials has been modeled to try and predict the effects of water freezing with composite materials.[19] As a widely used composite material, concrete is also an important material susceptible to freeze-fracture.[20]

Chemistry

Platinum coated surface of fracture face of glass reinforced plastic SEM Stereo 500x

Composite material substructure

Materials and colloid sciences use freeze-fracture techniques to investigate the nature of more complex substances. Even without the visualizing the atoms and molecules the shapes and textures of the interface between reacting substances will have an impact on how they behave and react with each other.[21]

Solder

Eutectic solders rely on the re-distribution of chemistry within the solder as they cool from liquid to solid. This can be taken advantage of to allow less toxic solders lower temperature solders that still bind well enough to prevent fracturing under operating conditions.[3]

Biology

The idea of looking at the detail of biological cells and proteins in detail, without chemical fixation, plastic or wax embedding and chemical staining prior to sectioning resulted in extensive use of freeze-fracture microscopy in biology.

Freeze-fracture-replication

The first documented systematic visualization a frozen, fractured surface to look at the structure of the fracture face itself was done in the mid-1950s. Russel L. Steere [22] was looking at virus particles and became concerned that conventional preparation techniques for electron microscopy, which included dehydration and more, may be altering the virus structure. While acknowledging freezing the sample would also cause changes he considered using it would give a different view. A "planed surface" was created on the frozen material using a knife. Due to the conditions required for the transmission electron microscope at the time the rapidly frozen and fractured virus itself could not be viewed directly. Instead Steere made a carbon re-enforced chromium replica of the fractured surface based on a procedure devised by others.[23] Steere overcame the problem of ice crystals forming on the fractured viruses by etching them away as done by others in 1955 prior to making the copy for viewing.[24] The additional etching step adds additional variations to the appearance of the original fractured surface but is essential to get rid of the condensates from the surface.

Commercialization of biological freeze-fracture

File:Freeze fracture under liquid nitrogen.webmthumb|Freeze-fracture Replication shielding block built in 1984 after Bullivant and Ames 1966thumb|Commercial Freeze Etching Replication device circa 2000 With the principles of visualizing a freeze-fractured surface for electron microscopy established, Moore automated and commercialized the Freeze-Fracture-Etch-Replication method in 1961 calling it "Steer's freeze-etching method". Believing a sharp knife was required to achieve a controlled fracture he used a microtome in a vacuum chamber to fracture the specimens.[25] Much cheaper, non-commercial alternatives which did not rely on a microtome or etching to clean the fracture face were later established. The technique "Freeze-Fracture Replication", rather than Steere and Moore's Freeze-Fracture Etching replication began to be used for the first time in this context. Adequate shielding from contamination in the vacuum required for replication meant etching was not required to clean the fracture face.[26] For the Bullivant & Ames method cheaply modified standard coating machines already routinely used in electron microscope laboratories, initially using a Meccano set. For smaller electron microsopy labs these could be more easily used than a large, specialized, commercial piece of freeze-fracture etch replication equipment. The method also allowed for much greater variations to the way the specimens could be fractured.[17]

Viewing inside membranes

During the 1960s-1980's it became apparent that the cell's lipid bilayer was shown to split into two halves revealing the interior when fractured under suitable conditions.[27]

The interior of a sheep lens cell plasma membrane. The smooth areas mainly at the top of the picture are mainly lipid membrane. The rough areas correlate to areas of protein in the membrane. When viewed closely the Freeze-fracture replica immunolabling (FRIL) reveals the gold beads averaging 5nm in size coated in anti-Cxn46 antibodies. They pepper the visible portion of the gap junction containing the protein[17]

Freeze-fracture was the only method to give a planar view of the membrane interior so some effort was needed to establish what aspects of a membrane interior image were native to the biology and which were produced by the freezing, fracturing and replication processes. Adequate explanations as to why the two fracture halves were not always complementary and why the fracture plane sometimes went between the lipid bilayer and not other times were found through the use of additional freeze-fracture techniques.[17] By 1989 the identity of "bumps" in the membrane as intramembrane protein particles was also established using a further modification of the basic freeze-fracture technique called freeze-fracture replica immunolabelling (FRIL).[28]

References

  1. ↑ 孙, 宝洋; 李, 占斌; 肖, 俊波; 张, 乐涛; 马, 波; 李, 建明; 程, 冬兵 (2019). "Research progress on the effects of freeze-thaw on soil physical and chemical properties and wind and water erosion". 应用生态学报 30 (1): 337–347. doi:10.13287/j.1001-9332.201901.019. PMID 30907557. 
  2. ↑ Liu, Hui; Guo, Xiao-Na; Zhu, Ke-Xue (September 2022). "Effects of freeze-thaw cycles on the quality of frozen raw noodles". Food Chemistry 387: 132940. doi:10.1016/j.foodchem.2022.132940. PMID 35429938. 
  3. ↑ 3.0 3.1 3.2 Chang, Fu-Ling; Lin, Yu-Hsin; Hung, Han-Tang; Kao, Chen-Wei; Kao, C. R. (22 April 2023). "Artifact-Free Microstructures in the Interfacial Reaction between Eutectic In-48Sn and Cu Using Ion Milling". Materials 16 (9): 3290. doi:10.3390/ma16093290. PMID 37176172. Bibcode: 2023Mate...16.3290C. 
  4. ↑ Bullivant, S. (September 1977). "Evaluation of membrane structure facts and artefacts produced during freeze‐fracturing". Journal of Microscopy 111 (1): 101–116. doi:10.1111/j.1365-2818.1977.tb00050.x. PMID 606830. 
  5. ↑ 5.0 5.1 Steere, R L; Erbe, E F; Moseley, J M (1 July 1980). "Prefracture and cold-fracture images of yeast plasma membranes.". The Journal of Cell Biology 86 (1): 113–122. doi:10.1083/jcb.86.1.113. PMID 6998983. 
  6. ↑ Lepault, J.; Dubochet, J. (1 August 1980). "Freezing, fracturing, and etching artifacts in particulate suspensions". Journal of Ultrastructure Research 72 (2): 223–233. doi:10.1016/S0022-5320(80)90060-X. PMID 7420536. 
  7. ↑ Sleytr, U. B.; Robards, A. W. (April 1982). "Understanding the artefact problem in freeze-fracture replication: a review". Journal of Microscopy 126 (1): 101–122. doi:10.1111/j.1365-2818.1982.tb00361.x. PMID 7040668. 
  8. ↑ Hmel, Peter J.; Kennedy, Anthony; Quiles, John G.; Gorogias, Martha; Seelbaugh, Joseph P.; Morrissette, Craig R.; Van Ness, Kenneth; Reid, T.J. (July 2002). "Physical and thermal properties of blood storage bags: implications for shipping frozen components on dry ice". Transfusion 42 (7): 836–846. doi:10.1046/j.1537-2995.2002.00135.x. PMID 12375655. 
  9. ↑ Fowler, Alex; Toner, Mehmet (March 2006). "Cryo‐Injury and Biopreservation". Annals of the New York Academy of Sciences 1066 (1): 119–135. doi:10.1196/annals.1363.010. PMID 16533923. 
  10. ↑ Nei, Tokio (March 1978). "Structure and function of frozen cells: freezing patterns and post-thaw survival". Journal of Microscopy 112 (2): 197–204. doi:10.1111/j.1365-2818.1978.tb01165.x. PMID 349159. 
  11. ↑ 11.0 11.1 Borgström, Henrik (8 April 2021). "Influence of Strain Rate, Temperature and Chemical Composition on High Silicon Ductile Iron". Minerals 11 (4): 391. doi:10.3390/min11040391. Bibcode: 2021Mine...11..391B. 
  12. ↑ González-Martínez, Rodolfo; Sertucha, Jon; Lacaze, Jacques (June 2023). "The mechanism of intermediate temperature embrittlement of cast irons by magnesium". Materials Today Communications 35: 106128. doi:10.1016/j.mtcomm.2023.106128. 
  13. ↑ Schorghofer, Norbert; Williams, Jean‐Pierre; Martinez‐Camacho, Jose; Paige, David A.; Siegler, Matthew A. (28 October 2021). "Carbon Dioxide Cold Traps on the Moon". Geophysical Research Letters 48 (20). doi:10.1029/2021GL095533. Bibcode: 2021GeoRL..4895533S. 
  14. ↑ Doute, S (December 1999). "Evidence for Methane Segregation at the Surface of Pluto". Icarus 142 (2): 421–444. doi:10.1006/icar.1999.6226. Bibcode: 1999Icar..142..421D. 
  15. ↑ Maynard-Casely, H. E.; Hester, J. R.; Brand, H. E. A. (1 September 2020). "Re-examining the crystal structure behaviour of nitrogen and methane". IUCrJ 7 (5): 844–851. doi:10.1107/S2052252520007460. PMID 32939276. 
  16. ↑ Andrews, E.H.. Fracture in polymers. Edinburgh and London: Oliver and Boyde. p. 204. 
  17. ↑ 17.0 17.1 17.2 17.3 Gruijters, W. T. M.; Bullivant, S. (March 1986). "Freeze‐fracturing at defined temperatures provides information on temperature rise during fracture, and on membrane complementarity". Journal of Microscopy 141 (3): 291–301. doi:10.1111/j.1365-2818.1986.tb02723.x. 
  18. ↑ Gomez, Jose´ P; Casto, Brian (1 January 1996). "Freeze-thaw durability of composite materials." (in English). https://rosap.ntl.bts.gov/view/dot/19417. 
  19. ↑ Nie, G. H.; Roy, Samit; Dutta, Piyush K. (December 2004). "Failure in Composite Materials due to Volumetric Expansion of Freezing Moisture". Journal of Cold Regions Engineering 18 (4): 135–154. doi:10.1061/(ASCE)0887-381X(2004)18:4(135). 
  20. ↑ Li, Wenhao; Hu, Shaowei (14 September 2023). "Fracture Behavior of Concrete under Chlorine Salt Attack Exposed to Freeze–Thaw Cycles Environment". Materials 16 (18): 6205. doi:10.3390/ma16186205. PMID 37763483. Bibcode: 2023Mate...16.6205L. 
  21. ↑ Zasadzinski, Joseph A. N.; Bailey, Stuart M. (December 1989). "Applications of freeze‐fracture replication to problems in materials and colloid science". Journal of Electron Microscopy Technique 13 (4): 309–334. doi:10.1002/jemt.1060130406. PMID 2681575. 
  22. ↑ Steere, Russell L. (25 January 1957). "Electron Microscopy of Structural Detail in Frozen Biological Specimens". The Journal of Cell Biology 3 (1): 45–60. doi:10.1083/jcb.3.1.45. PMID 13416310. 
  23. ↑ Williams, R. C.; Wyckoff, R. W. G. (1 March 1945). "Electron Shadow-Micrography of Virus Particles.". Experimental Biology and Medicine 58 (3): 265–270. doi:10.3181/00379727-58-14918. 
  24. ↑ Meryman, H.T.; Kafig, E. (1955). "The study of frozen specimens, ice crystals and ice crystal growth by electron microscopy.". Naval Medical Research Institute, National Naval Medical Center.. 
  25. ↑ Moor, H.; Mühlethaler, K.; Waldner, H.; Frey-Wyssling, A. (1 May 1961). "A New Freezing-Ultramicrotome". The Journal of Cell Biology 10 (1): 1–13. doi:10.1083/jcb.10.1.1. PMID 13772269. 
  26. ↑ Bullivant, Stanley; Ames, Adelbert (1 June 1966). "A Simple Freeze-Fracture Replication Method for Electron Microscopy". The Journal of Cell Biology 29 (3): 435–447. doi:10.1083/jcb.29.3.435. PMID 5962938. 
  27. ↑ Branton, Daniel; Bullivant, Stanley; Gilula, Norton B.; Karnovsky, Morris J.; Moor, H.; Mühlethaler, K.; Northcote, D. H.; Packer, Lester et al. (3 October 1975). "Freeze-Etching Nomenclature". Science 190 (4209): 54–56. doi:10.1126/science.1166299. PMID 1166299. Bibcode: 1975Sci...190...54B. 
  28. ↑ Gruijters, W T; Kistler, J; Bullivant, S; Goodenough, D A (1 March 1987). "Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions.". The Journal of Cell Biology 104 (3): 565–572. doi:10.1083/jcb.104.3.565. PMID 3818793. 



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