Figure 1 depicts a schematic of our SR microscope, which was built around an Olympus IX71 inverted base and equipped with multiple laser lines and shutters to accommodate PALM, STORM, and dSTORM imaging modes. A solid-state violet laser (405 nm, CrystaLaser), sapphire blue laser (488 nm, Coherent), and diode-pumped green laser (561 nm, CrystaLaser) were coaligned using appropriate dichroic beamsplitters (Semrock LM01-427 and LM01-503, Rochester, New York). As illustrated for the 488-nm laser, the collimated excitation beam was directed by a multilaser line excitation dichroic (Semrock Di01 R405/488/561/635) and focused onto the back aperture of a 1.49 NA oil-immersion objective (Olympus). In order to preferentially excite restructuring actin several microns away from the cover glass surface, we utilized Hi-Lo microscopy,31 which reduces background considerably compared to epi-excitation. This was achieved by translating the 488-nm laser toward, but not to, the critical angle required for total internal reflection (TIR). Since TIR conditions were not met, the excitation beam exited the objective at a very low angle. The final angle of the outgoing beam was fine tuned for each sample to maximize the signal-to-noise ratio. Fluorescence emission was collected through the same objective, spectrally filtered, and then focused onto the active area of an electron-multiplying (EM) CCD camera (Princeton Instruments, Trenton, New Jersey) Pro-EM, ). For these experiments, the standard tube lens was removed from the microscope and a 450-mm achromat (Edmund Optics 49-282, Barrington, New Jersey) was used in its place. The objective-achromat optical relay resulted in a total magnification of and mapped each pixel of the EM-CCD onto a area in sample space, with this pixel size chosen to optimize localization precision following the estimates of Thompson et al.12 To eliminate Z-drift during image acquisition, we employed a custom autofocus module that used a 635-nm diode laser beam (Thorlabs LDM635, Newton, New Jersey) sent into the microscope in through-objective TIR mode on the opposite side of the objective from the 488-nm excitation beam. The returning 635-nm beam was focused onto the active area of a split photodiode and the voltage outputs were sent into a PCI I/O board (National Instruments, Austin, Texas) and used as inputs to a proportional-integral-differential (PID) feedback loop that adjusted the Z position of the objective through a piezo controlled microscope objective mount (P-725 PIFOC, Physik Instrumente, Auburn, Massachusetts). The goal of the PID feedback loop was to keep the voltages on the split photodiode equal, thus maintaining a constant focus on the EM-CCD camera during image acquisition. The entire instrument (shutters, EM-CCD acquisition, Z autofocus system) was controlled and automated using custom LabVIEW programs.