Single Pixel Reconstruction Imaging: taking confocal imaging to the extreme

Abstract

Light nanoscopy is attracting widespread interest for the visualization of fluorescent structures at the nanometre scale. Recently, a variety of methods have overcome the diffraction limit, yet in practice they are often constrained by the requirement of special fluorophores, nontrivial data processing, or a high price and complex implementation. Therefore, confocal microscopy, yielding a relatively low resolution, is still the dominant method in biosciences. It was shown that image scanning microscopy (ISM) with an array detector could improve the resolution of confocal microscopy. Here, we review the principles of the confocal microscopy and present a simple method based on ISM with a different image reconstruction approach, which can be easily implemented in any camera-based laser-scanning set-up to experimentally obtain the theoretical resolution limit of confocal microscopy. Our method, single pixel reconstruction imaging (SPiRI), enables high-resolution 3D imaging utilizing image formation only from a single pixel of each of the recorded frames. We achieve the experimental axial resolution of 330 nm, which was not shown before by basic confocal or ISM-based systems. The SPiRI method exhibits a low lateral-to-axial FWHM aspect ratio, which means a considerable improvement in 3D fluorescence imaging. As a demonstration of SPiRI, we present the 3D-structure of a bacterial chromosome with an excellent precision.