Improvement in Resolution of Multiphoton Scanning Structured Illumination Microscopy via Harmonics

Abstract

We describe a multiphoton (mP)-structured illumination microscopy (SIM) technique, which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence. This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal. The harmonics of the structured fluorescence illumination are utilised to improve resolution. We present an mP-SIM theory for reconstructing the super-resolution image of the system. Theoretically, the resolution of our mP-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered. Experimentally, we demonstrate an 86 nm lateral resolution for two-photon (2P)-SIM and a 72 nm lateral resolution for second-harmonic-generation (SHG)-SIM. We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon. Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.