Question

(1 mark) 2. What is chemical fixation and why is fixation important when preparing biological samples? (2 marks) 3. Describe the main steps involved in preparing a sample for transmission electron microscopy. (2 marks) 4. Describe how an image is formed with a transmission electron microscope. (2 marks) 5. Describe the main steps involved in preparing a sample for scanning electron microscopy. (2 marks) 6. Describe how an image is formed with a scanning electron microscope. (2 marks) 7. Briefly describe the process of freeze fracturing. (2 marks) e here to search O e 9 4 DI Priser Home ග do PA > % 5 & 7 9 0 8 2 3 4 > W E R T U S D F G H K'S N. M

Answer 1

2. Fixation is a technique used to preserve biological tissues or samples. There are three types of tissue fixation, heat fixation, immersion and perfusion.  Chemical fixatives are used in immersion (Immersing the tissue specimen in a chemical fixative) and perfusion (used to fix small animals or a whole organ via blood flow) to preserve tissue specimens. Fixation is an important step in histology for the preservation of tissue specimens to avoid further biochemical reactions and increase the stability. It protects the tissues and tissue components from decay and subsequent processing.

3. Sample preparation for TEM (Transmission electron microscopy)

• Fix the specimen either by chemical fixation of cryopreservation for stabilizing the cells so that the cells will not damage.
• Wash the tissue specimen with sodium cacodylate buffer to maintain pH.
• The tissue specimen is again fixed by osmium tetroxide (OsO4) to increase the contrast.
• Stain the sample and dehydrate the specimen with any organic solvents (ethanol or acetone) to remove water contents.
• Infiltrate the tissue with epoxy resin to embede and polish the specimen to avoid scratches.
• Cut the sample into 30mmx60mm using a microtome and fix on the glass slide with a semitransparent thermoplastic glue to achive semitransparent nature (The TEM sample must be semitransparent).
• Stain the sample again after sectioning.
• Place the tissue specimen into the sample holder.

4. The TEM transmits a beam of electrons through a specimen through a hole in the anode plate and encounter the condenser lenses and then ojjective lenses surrounded the specimen and to the specimen port and produce an image of an object. The image of the specimen is magnified and focused on to a fluorescent screen with a high resolution.

5. In Scanning electron microscopy (SEM), first fix the sample with aldehydes and again with osmium tetroxide to prevent extraction. The specimen is then dehydrated with organic solvents, such as ethanol or acetone to remove water contents. Dry the specimen by allowing to evaporate the organic solvents. Mount the specimen on a metal stub with sticky carbon disc and do sputter coating with conductive material (gold).