BIOCHEMICAL STUDIES 



AT THE ELECTRON MICROSCOPE LEVEE 



ON STRUCTURALLY INTACT 



FREEZE-DRIED SUBCELLULAR ORGANELLES 



B. W. GRUNBAUM 



Cancer Research Institute, 



University of Califomia Médical Center, 



San Francisco 



Pancréas, liver, heart muscle and meimmary tissues were freeze-dried in a 

 specially constructed apparatus which permitted préservation of tissue 

 structure for biocheraical and cytological studies at the électron micro- 

 scope level without the use of chemical fixatives and stains. The design 

 of the apparatus incorporâtes a number of features which facilitate its 

 opération and produce good results: (1) the tissue is embedded in metha- 

 crylate or paraffin within the drying chamber without breaking the vacuum; 

 (2) vacuum of 10"* to 10"* mm H g pressure is easily obtained within 15 

 minutes because the vacuum System, except the drying chamber, is pre- 

 -evacuated; (3) the température of the tissue after quenching below -160°C 

 does not rise above -70°C until dry; (4) once the spécimen is placed in an 

 open gelatin capsule, which acts as a mould for the embedding material, 

 the tissue is not handled until it is ready for sectioning. Sections eut at 

 1/20 of a micron revealed hi^ contrast of subcellular détail when viewed 

 with the électron microscope. It was possible to identify fat and protein 

 particles ranging in size from 500 to 8000 m^ and 40 to 300 mpt respectively 

 in the lactating mammary gland. Biochemical studies were carried out 

 directly on the électron microscope grids carrying the spécimen. Morpho- 

 logically the freeze-dried tissues differed from those fixed in osmium- 

 -tetroxide. Slides will be shown of the freeze-drying apparatus and various 

 tissue sections as seen with the light and électron microscopes. The 

 technique of biochemical analysis of mammary tissue and its gênerai 

 application will be discussed. 



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