B.

Sc (P) Life Science III year Semester VI

DSE-1 Analytical Techniques in Plant Sciences
Practical- Study of Microscopic Techniques-1
Dr Sarla, Dr DK Mallick & Dr Madhu Rani, Department of Botany

Freeze Fracture Technique

▪ The freeze fracture technique was developed by Hans Moor in 1961.
▪ Freeze-fracture technique is a process in which biological specimens or nanomaterial are frozen, fractured,
and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron
microscopy.
▪ The specimens are subjected to ultra-rapid freezing immobilizing the cellular components immediately.
▪ The freezing is carried out in the presence of cryoprotective agents such as glycerol to prevent ice crystal
formation.
▪ The frozen specimen are then fractured at liquid nitrogen cooled temperatures under high vacuum. The
fractured specimen splits into two halves conferring two faces called by convention the PF-face (plasma
fracture-face) and EF-face (extracellular fracture-face).
▪ The fracture is irregular and occurs along lines of weakness like the plasma membrane or surfaces of
organelles.
▪ The resultant fractured surface is then shadowed with platinum vapor from an angle that confers three-
dimensional detail to the cast. This is followed by evaporation of carbon vertically onto the surface to
produce carbon replica.
▪ The organic material is digested away during the procedure leaving a replica.
▪ The carbon-metal replica is trimmed to proper size retrieved onto a standard EM grid and examined under
electron microscope. The replica is actually a template like impression of the distribution of particulars in
the original specimen.
▪ Freeze-fracture preparations are examined by transmission electron microscopy and their major
contribution to high resolution morphologic studies is their unique representation of structure/function
elements of cell membranes.
▪ The electron beam readily passes through the portions of the replica containing the carbon but is absorbed
by the areas containing platinum.
▪ The resulting images which have a three-dimensional impact are considered different from those obtained
with sectional materials.
▪ The shadowing angle used in preparation of the replica is usually indicated in the resulting EM micrograph
(the EM should be viewed along the shadowing angle otherwise depressions on the surface may be seen
as projections or vice versa).
▪ This technique is used specially for the investigation of cell membranes and their specializations and has
contributed considerably to the understanding of cellular form to related cell function.
▪ This technique has also been widely used for ultrastructural investigation in many areas of cell biology
and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials
science studies.

MICROSCOPIC TEHNIQUES SG, DM, MR

The Freeze-Fracture Technique. In steps (a) and (b), a frozen eucaryotic cell is fractured with a cold knife. Etching
by sublimation is depicted in (c). Shadowing with platinum plus carbon and replica formation are shown in (d) and (e).

Freeze Fractured view of interiors of a Chlorella cell.

https://doi.org/10.1038/nprot.2007.55

MICROSCOPIC TEHNIQUES SG, DM, MR

SOURCES
Carson, J.L. Fundamental Technical Elements of Freeze-fracture/Freeze-etch in
Biological Electron Microscopy. J. Vis. Exp. (91). (2014)
Lansing M Prescott, Donald A Klein and John P Harley. Microbiology, 5/e. McGraw Hill
Publications (2002).
Severs, N. Freeze-fracture electron microscopy. Nat Protoc 2, 547–576 (2007).
Black, Jacquelyn G. Microbiology: principles and explorations. 8th ed. John Wiley &
Sons Inc. (2012)

MICROSCOPIC TEHNIQUES SG, DM, MR