Structured‐illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in‐principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency–space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two‐dimensional (2D) illumination patterns or sequentially rotated one‐dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal‐to‐noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background.
Structured‐illumination microscopy (SIM) is a high‐resolution light microscopy technique that allows imaging of fluorescence at a resolution about twice the classical diffraction limit. There are various ways that the illumination can be structured, but it is not obvious how the choice of illumination pattern affects the final image quality, especially in view of the noise. We present a detailed performance analysis considering two illumination techniques: sequential illumination with line‐gratings that are shifted and rotated during image acquisition and two‐dimensional (2D) illumination structures requiring only shift operations. Our analysis is based on analytical theory, supported by simulations of images considering noise. We also extend our analysis to a nonlinear variant of SIM, with which enhanced resolution can be achieved, limited only by noise. This includes nonlinear SIM based on the light‐induced switching of the fluorescent molecules between a bright and a dark state. We find sequential illumination with line‐gratings to be advantageous in ordinary (linear) SIM, whereas 2D patterns provides a slight signal‐to‐noise advantage under idealised conditions in nonlinear SIM if there is no nonswitching background.
