Preparation of a Selected Cell for Electron Microscopy

In the course of a study of the effects of micro-beam irradiation of a small portion of a particular cell in culture, it became necessary to devise a technic for following this cell through the processes of fixation and subsequent embedding for electron microtomy. The new procedure has proved more satisfactory for our purposes than the methods previously described by Borysko and Sapranauskas (1), Gay (2), Nebel and Minick (3), and Howatson and Almeida (4). The cultures are made on glass or quartz coverslips }~ inches in diameter. Quartz is necessary if the cell is to be irradiated with the Uretz ultraviolet microbeam (5). With a diamond marker, a circle is made on the coverslip around the selected cell, which is thus easily found thereafter. The culture is fixed either by introducing a volatile agent-as iodine, formaldehyde, or osmium te-troxide-into the culture chamber or by floating the coverslip, culture side down, on the fixing solution. As the cell is flattened on the coverslip, the time required for fixation is shorter than for blocks of tissue. The coverslip with culture down is then floated on water and subsequently passed by immersion, culture side up, through progressive concentrations of alcohol from 20 to 100 per cent preliminary to embedding. Any vaseline on cover-slip must be removed. The following procedure, used for embedding most of the cells, is a modification of the usual technic for embedding tissues in nitrocellulose for light microscopy. Among other advantages, the method is carried out at room temperature. After dehydration, the culture, still on the cover-slip, is passed through a solution of equal parts of absolute alcohol and ethylene dichloride and then through ethylene dichloride alone. On removal from this solvent, the wet culture is immediately covered with a fairly large viscous drop of a polymerized mixture of 1 part methyl and 4 to 9 parts of butyl methacrylate in ethylene dichloride. The solvent is then allowed to evaporate slowly from the culture in a covered dish. After some hours the plastic is quite firm and cuts easily. Of the other solvents tried, only benzol gave results as good as ethylene dichloride except when it was used for osmium-fixed tissue. Some cultures were infiltrated with a solution of the polymerized methacrylates in their mono-mers and then polymerized with ultraviolet light. The results so far have not been so good as with ethylene dichloride. A few attempts were made …