piSTORM: programmable illumination in Stochastic Optical Reconstruction Microscopy

We implement a programmable illumination system in stochastic optical reconstruction microscopy (STORM) using holographic modulation of the excitation beam profile in the sample plane of a widefield fluorescence microscope. A binary hologram is implemented in a plane conjugate to the sample plane using a liquid crystal spatial light modulator (LCSLM). This approach allows a user-defined yet complex illumination pattern to match approximately any arbitrary region of interest (ROI) within the field of view (FOV) of the microscope, while keeping regions outside the ROI unilluminated. The proposed programmable illumination scheme minimizes unnecessary photobleaching of areas not overlapped with the arbitrary illumination pattern, which can be changed in real time programmably without the need for any mechanical movement. The scheme thus facilitates super-resolved STORM imaging, at different instants, of different ROIs of arbitrary shapes in the same FOV of the widefield microscope. We present here a successful experimental implementation of the scheme to image quantum dots and closely spaced actin filaments in human brain cancer (U87MG) cells.