Functionalization of the upper cantilever surface with bR membrane patches visualized by tapping mode AFM. The scale bar corresponds to 1 μm. The dashed line, also indicated by two arrowheads in panel A, corresponds to the position of the captured height profile (B). (C) Nonlabeled bR membrane patches immobilized on ultraflat gold (in air, tapping mode). (D) Immunoassayed bR patches. Antibodies are specific against the extracellular side of bR, indicating a preferential orientation of bR with the cytoplasmatic side facing the cantilever. Scale bar, 500 nm.

Deflection measurement of bR-functionalized cantilevers (solid circles) versus blank gold cantilevers (open circles). From the 6574 data points, only 30 are labeled and attributed with an error bar indicating the standard deviation of the averaging (three deflection measurements each). Section I, buffer equilibration for baseline; Section II, incubation time with 200 mM hydroxylamine; Section III, after rinsing with buffer. To obtain normalized deflection values (in nanometers), the deflection was first divided with the peak height of the heat test and then multiplied with the average peak height (see Methods).

Schematic diagram of the setup. bR 2D crystals are immobilized on the upper surface of the cantilever. The deflection of the cantilever is optically detected with a laser using a PSD. To bleach the bR molecules in situ, an LED with an emission maximum at 565 nm was placed above the cantilever. Note that the cystein of the G241C mutant was not essential for membrane anchoring and orientation (see Discussion).

Prebleaching of bR crystal before immobilization on the cantilever. The spectra were normalized at 280 nm and the prebleaching grade was determined at 568 nm. Open circles: unbleached; open squares: 33% bleached; filled circles: 66% bleached; and filled squares: 100% bleached. The latter was used to functionalize the reference cantilever.

(A) Differential measurement of cantilever deflection with 100% prebleached bR as reference. The gray area (section II) indicates the injection of hydroxylamine and incubation time where interpretation of the data is complicated by some unspecific interactions (see Discussion). The short gray lines indicate the slopes depicted in panel B by linear regression. (B) Initial slope after buffer injection versus the prebleaching grade of bR before cantilever functionalization. The slopes were determined between time point 220 and 222 (A). The error bars represent the estimated standard deviation of the slope determination. The black line represents a linear regression of the data (Pearson coefficient: R = −0.99288). (○) Unbleached; (□) 33% bleached; (•) 66% bleached; (▪) 100% bleached (reference in panel A).