Methods of Preparation for Electron Microscopy: An by Prof. David G. Robinson Ph.D., Priv. Doz. Dr. Ulrich Ehlers,

By Prof. David G. Robinson Ph.D., Priv. Doz. Dr. Ulrich Ehlers, Prof. Dr. Rainer Herken, Prof. Dr. Bernd Herrmann, Prof. Dr. Frank Mayer, Prof. Dr. Friedrich-Wilhelm Schürmann (auth.)

In 1939, while the electron optics laboratory of Siemens & Halske Inc. started to manufacture the 1st electron microscopes, the organic and clinical profes­ sions had an unforeseen tool at their disposal which surpassed the reso­ lution of the sunshine microscope by means of greater than a hundredfold. The speedy and huge program of this new software used to be complex through the overpowering prob­ lems inherent in specimen practise for the research of mobile struc­ tures. The microtechniques utilized in mild microscopy have been now not appli­ cable, on account that even the thinnest paraffin layers couldn't be penetrated by means of electrons. Many powerfuble organic and scientific learn staff expressed their anxiousness that gadgets in excessive vacuum will be converted as a result of entire dehydration and the absorbed electron strength may finally reason degrada­ tion to rudimentary carbon backbones. It additionally appeared questionable as to if it might be attainable to arrange skinny sections of roughly zero. five 11m from heterogeneous organic specimens. hence one used to be without warning in posses­ sion of a unique software which, compared to the sunshine microscope, allowed a 10-100-fold better answer, but an appropriate instruction method was once missing. This sceptical perspective in the direction of the appliance of electron microscopy in bi­ ology and medication was once supported at the same time via the overall opinion of colloid chemists, who postulated that during the submicroscopic quarter of dwelling buildings no solid development blocks existed that may be printed with this apparatus.

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Body fluids, tissues and cells can have different osmotic values depending on the animal and its particular physiological condition. Since the osmotic pressure of the fixation solution can considerably influence structural integrity, it is important to appropriately adjust the osmolarity of the fixation solution. As a guide, the osmolarity of the fixation solution should correspond to that of the body fluid or blood, i. e. it should be iso- or slightly hypertonic. Solutions which are too hypertonic lead to shrinkage and deformation of the organ or tissue in question; solutions which are too hypotonic lead, in contrast, to a swelling.

However, cells fixed with OS04 are no longer osmotically active; their membranes lose their differential permeability properties. 2 Aldehydes. Of the various aldehydes which are employed as fixatives in EM glutar(di)aldehyde (C sH g0 2) is the most important. Acrolein (C3 H sO) and paraformaldehyde (CH 20) are also in use, particularly when a rapid penetration of the fixative is required. They all stabilize proteins by creating interand intrachain cross links. As a result of aldol condensations, several glutaraldehyde molecules can be linked together between neighbouring amino acid chains.

G. to the main axis of the body. In cutting the tissue (with a new, sharp razor blade) it is important not to apply too much pressure - the razor blade should be pulled gently through the tissue. The arm of a pair of fine tweezers can be used as a guide for this cutting action, which should be carried out in liquid under a bin- The Fixation of Animal Cells 37 ocular microscope. For particularly sensitive material the tissue can be embedded in agarose (3%, w/v, warmed to 45° C then allowed to cool after insertion of the tissue) and then cut into blocks (see also Andres and von During 1981).

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