Wednesday, 10 February 2016

Hexamethyl Disilazane as an Alternative to Critical Point Drying

Critical point drying (CPD) is an established method of dehydrating biological tissue prior to examination in the Scanning Electron Microscope (SEM). This technique was first introduced to preserve three-dimensional structure of biological specimens for transmission electron microscopy, later it was used for obtaining dry specimens for SEM examination. Although CPD is a well-established method for drying specimens, there has been compelling evidence that it can damage soft specimens in two different ways: Structural integrity and Chemical Integrity. Over the years, use of the pharmaintermediate, Hexamethyl Disilazane (HMDS) in critical point drying is an established method of dehydrating biological tissue prior to examination in the SEM. Moreover, use HMDS in the imaging of cells on coal instead of CPD have led to negating the need for expensive equipment.
Studies on the applications of Hexamethyl Disilazane (HMDS) over Critical Point Drying (CPD):

• CPD and HMDS sample preparation techniques were used for testing the effects of both on the cervical cells on field emission scanning electron microscopy and energy dispersive X-ray. The results indicated that SEM imaging, elemental composition, and processing time for sample preparation with the HMDS technique were better than CPD technique for cervical cell preparation technique because in terms of weight percentages of carbon and oxygen element compositions in HMDS technique were higher than the CPD technique.

• CPD and HMDS samples used for preserving biological structures, revealed that samples prepared using CPD had tissue damage in the form of cracking. In addition there was excess amount of debris around the crack openings. Another potential result that placed HMDS as a superior drying agent is the time constraints that can be effective parameters while preparing more specimens; use of the Pharma Intermediate may be preferred over the traditional technique as it allows multiple samples to be dried simultaneously.

Preventing the gas/liquid interface is the main goal of critical point drying (as well as the alternative process of HMDS). Whenever a liquid evaporates into the gaseous phase, large surface tension phenomena occur. These surface tensions can harm fine surface details on the surface of the sample. CPD usually takes up to 3 hours to perform. Using Hexamethyldisilazane instead of CPD is quicker and can yield acceptable results on some samples. When the sample is dehydrated to 100% ethanol, a fifty/fifty mix of ethanol/HMDS can be placed on the sample followed by 2 or 3 exchanges of HMDS. After these exchanges, sample is in a fume hood where HMDS evaporates off. The vapor pressure of HMDS is such that surface damage is minimal.

The pharmaintermediate, Hexamethyl Disilazane is extensively used to replace the critical drying technique in sample preparation of tardigrades for SEM imaging because conventional procedures required CPD apparatus or machines to achieve the suitable temperature/pressure combination to completely dehydrate specimens, but with HMDS, the specimens can be dehydrated by simply bathing them in HMDS, ethanol solution, eliminating the use of any special equipment. All in all, the use of HMDS in SEM instead of CPD is preferred as it is safer, cheaper and more practical.