Artifact analysis and fast image reconstruction for spinning disk confocal Image Scanning Microscopy

Abstract

Image Scanning Microscopy is a linear super-resolution fluorescence microscopy, which does not involve very complex optical system design, but is based on existing setup and capable of taking the advantage of computing and image processing methods to achieve super-resolution imaging. As ISM image acquisition and image reconstruction are actually based on discrete space, some factors, such as the pattern of scanning path, reconstruction accuracy, and background could result in artifacts, which could affect the image-quality of the final reconstructed ISM image seriously and has to be taken into consideration in practice. In this article, we analyzed the artifact problem frequently emerged in the spinning disk confocal (SDC) ISM systems, based on which a modified ISM reconstruction technique is proposed to accelerate the image reconstruction and avoid potential artifacts. The experimental result shows that the proposed ISM reconstruction method can be 10 times faster than the existing method and it validates that artifacts can be caused by multiple factors, i.e. the inhomogeneity of sampling-interval for scanning, pixels reassignment accuracy and the background. It also shows that these artifacts can be significantly reduced by these steps: remove image background from the raw image, calculate ISM image with accurate reconstruction and apply appropriate deconvolution to the accurately reconstructed ISM image. The proposed ISM image reconstruction and the artifacts removal technique improves the image reconstruction significantly and enable SDC-ISM system to guarantee high image-quality output.