Mutifocal image scanning microscopy based on double-helix point spread function engineering

Abstract

Confocal laser scanning microscopy (CLSM) is a powerful imaging tool providing high resolution and optical sectioning. In its standard optical configuration, a pair of confocal pinholes is used to reject out-of-focus light. The diffraction limited resolution can be broken by reducing the confocal pinhole size. But this comes at the cost of extremely low signal-to-noise ratio (SNR). The limited SNR problem can be solved by image scanning microscopy (ISM), in which the single-point detector of a regular point-scanning confocal microscopy is substituted with an array detector such as CCD or CMOS, thus the two-fold super-resolution imaging can be achieved by pixel reassignment and deconvolution. However, the practical application of ISM is challenging due to its limited image acquisition speed. Here, we present a hybrid microscopy technique, named multifocal refocusing after scanning using helical phase engineering microscopy (MRESCH), which combines the double-helix point spread function (DH-PSF) engineering with multifocal structured illumination to dramatically improve the image acquisition speed. In the illumination path, sparse multifocal illumination patterns are generated by a digital micromirror device for parallel imaging information acquisition. In the detection path, a phase mask is introduced to modulate the conventional PSF to the DH-PSF, which provides volumetric information, and meanwhile, we also present a digital refocusing strategy for processing the collected raw data to recover the wild-filed image from different sample layers. To demonstrate imaging capabilities of MRESCH, we acquire the images of mitochondria in live HeLa cells and make a detailed comparison with those from the wide-field microscopy. In contrast to the conventional wide-field approach, the MRESCH can expand the imaging depth in a range from –1 μm to 1 μm. Next, we sample the F-actin of bovine pulmonary artery endothelial cells to characterize the lateral resolution of the MRESCH. The results show that the MRESCH has a better resolution capability than the conventional wide-field illumination microscopy. Finally, the proposed image scanning microscopy can record three-dimensional specimen information from a single multi-spot two-dimensional scan, which ensures faster data acquisition and larger field of view than ISM.