Holographic projection for multispot structured illumination microscopy

This paper describes the design and testing of a multispot structured illumination microscopy system using computer-generated holograms to create the required excitation patterns. Furthermore, it demonstrates the use of an adapted direct search algorithm for calculating the holograms that allows for imaging across an extended field of view. The system was tested on fixed targets and live cells yielding a two times resolution increase over conventional diffraction-limited imaging. LAY DESCRIPTION: We present the design and testing of a multispot structured illumination microscopy system using computer-generated holograms to create the required excitation patterns. It demonstrates the use of an adapted direct search algorithm for calculating the holograms that allows for imaging across an extended field of view. The system was tested on fixed targets and live cells yielding a two times resolution increase over conventional diffraction-limited imaging. The results here demonstrate that holography provides an efficient means of pattern projection for MSIM imaging. It provides a significant improvement in the efficiency of pattern projection and more importantly it allows for the testing of more diverse excitation patterns than possible with amplitude-only projection. For example, PSF engineering using phase modulation can be easily incorporated into the calculated holograms, potentially generating subdiffraction structures in the excitation pattern. The ability to incorporate PSF engineering into SIM opens up holographic MSIM as a potential method for further increasing resolution with little or no change to the imaging system.