Figure 1.

Summary of data collection and methods. (A) Block diagram of the microscope used in this work. Custom PC software controls all microscope functions and finalizes data acquisition. After scan parameters are entered, the scan program is sent to a field-programmable gate array (FPGA) as a metalanguage string of hexadecimal characters and saved into onboard memory. A Start command is sent with the number of repeats to begin a scan by driving a direct digital synthesis (DDS) board, producing a series of frequencies and chirp rates directing the acquisition of the volume, while a concurrent trigger signal is sent to the data acquisition oscilloscope. A laser diode (LD) is directed by the acousto-optic deflectors (AOD), through a telescope tube (TT), and reflected by a dichroic mirror (DM) onto the back-aperture of an objective (OBJ). The light emitted by the sample is collected on a photomultiplier tube (PMT) and converted into an image on the PC. (B) Example image from the microscope. Note that high and low concentrations, as seen here, are common, and that all trajectories are taken from single molecules that never overlap. (Dashed line) The z cut shown to the right. (C–E) Example trajectories of a bead in a (C) buffer solution; (D) extract; and (E) extract treated with nocodazole.