Confocal structured illumination microscopy for improving the signal-to-noise ratio and depth of fluorescent optical section imaging

Abstract

Light scattering from the sample is an unavoidable problem in fluorescence imaging. Compared with laser scanning confocal scanning microscopy, although optical-sectioning structured illumination microscopy (OS-SIM) has the advantages of fast imaging speed and low phototoxicity, it faces the challenge of removing the scattering fluorescent noise particularly when imaging thick and densely labeled sampling. To improve the imaging performance of OS-SIM, we introduce the concept of confocal imaging to OS-SIM and propose confocal structured illumination microscopy (CSIM). CSIM exploits the principle of dual imaging to reconstruct a dual image from each camera pixel. The scattered fluorescent noise and the unscattered fluorescent signal recorded by the camera pixel are separated in the reconstructed dual image. By extracting the unscattered fluorescent signal from each dual image based on the conjugate relationship between the camera and the spatial light modulator, we can eliminate the scattered fluorescent noise and reconstruct a confocal image. We have built the theoretical framework of CSIM. Experimental results of fluorescent optical-sectioning demonstrate that CSIM achieves a superior performance in eliminating scattered fluorescent noise and in extending imaging depth compared with existing OS-SIM. CSIM is expected to broaden the application range of OS-SIM.