THY-1 IMMUNOLABELED THYMOCYTE MICRODOMAINS STUDIES WITH THE ATOMIC-FORCE MICROSCOPE AND THE ELECTRON-MICROSCOPE

The atomic force microscope (AFM) and the transmission electron micros cope (TEM) have been used to study the morphology of isolated mouse th ymocyte microdomains and Thy-1 antigen distribution at the surface of these structures. AFM images were recorded in air in the contact mode on membrane Vesicles deposited on previously heated tissue culture pla stic sheets and indirectly immunolabeled for Thy-1 expression with col loidal gold-conjugated secondary antibodies. AFM images of untreated p lastic plates showed a very characteristic network of streaks 20-200 n m wide. Heating the plastic removed the streaks and provided flat surf aces (r.m.s. 1 nm). This substrate allowed strong adsorption and homog eneous spreading of the vesicles and easy manipulations during immunol abeling experiments. Vesicles flattened on the substrate without losin g their morphology. The 10-nm membrane-bound gold beads were reproduci bly imaged without degradation by repeated tip scanning. The observed microdomains had a mean diameter of 184 +/- 76 nm, and 65% of them wer e specifically labeled. Images obtained with the TEM on the same vesic les, deposited on carbon-coated grids and negatively stained, confirme d the AFM observations. The size distribution of the microdomains was quite similar, but the number of beads per vesicle was significantly h igher, and 76% of the vesicles were labeled. The difference may be exp lained 1) by removal of beads from the vesicles in the additional wash ing step with water, which was necessary for the AFM; 2) by tip-sample convolution; and 3) by statistical fluctuations.