To solve extended acquisition time issues inherent in the conventional hopping-scanning mode of scanning ion-conductance microscopy (SICM), a new transverse-fast scanning mode (TFSM) is proposed. Because the transverse motion in SICM is not the detection direction and therefore presents no collision problem, it has the ability to move at high speed. In TSFM, the SICM probe gradually descends in the vertical/detection direction and rapidly scans in the transverse/nondetection direction. Further, the highest point that decides the hopping height of each scanning line can be quickly obtained. In conventional hopping mode, however, the hopping height is artificially set without a priori knowledge and is typically very large. Consequently, TFSM greatly improves the scanning speed of the SICM imaging system by effectively reducing the hopping height of each pixel. This study verifies the feasibility of this novel scanning method via theoretical analysis and experimental study, and compares the speed and quality of the scanning images obtained in the TFSM with that of the conventional hopping mode. The experimental results indicate that the TFSM method has a faster scanning speed than other SICM scanning methods while maintaining the quality of the images. Therefore, TFSM provides the possibility to quickly obtain high-resolution three-dimensional topographical images of extremely complex samples.

Fluorescence resonance energy transfer (FRET) measurement based on partial acceptor photobleaching (PbFRET) is easy to implement without external references. However, the current PbFRET methods are inapplicable to the construct with multiple acceptors, which largely increase the Förster distance. Here, we proposed a linear theory for the dependence of the acceptor photobleaching probability of construct with multiple acceptors on the photobleaching degree (x) and developed a multiple acceptors PbFRET method (Ma-PbFRET) to measure the FRET efficiency of construct with multiple acceptors (n) by measuring the fluorescence intensities of both donor and acceptor channels before and after acceptor photobleaching. The Ma-PbFRET method was validated by measuring the FRET efficiency of construct with two or three acceptors under different x in living cells. Our experimental results demonstrate that the Ma-PbFRET method is capable of exactly quantifying the FRET efficiency of construct with multiple acceptors, providing a simple and powerful tool to investigate the assembly/disassembly of biomolecular complexes with larger distance in living cells.