Single Pixel Reconstruction Imaging: taking confocal imaging to the extreme

Light nanoscopy is attracting widespread interest for the visualization of fluorescent structures at the nanometer scale, especially in cellular biology. To achieve nanoscale resolution, one has to surpass the diffraction limit--a fundamental phenomenon determining the spot size of focused light. Recently, a variety of methods have overcome this limit, yet in practice they are often constrained by the requirement of special fluorophores, nontrivial data processing, or high price and complex implementation. For this reason, confocal fluorescence microscopy that yields relatively low resolution is still the dominant method in biomedical sciences. It was shown that image scanning microscopy (ISM) with an array detector instead of a point 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 the 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 experimental axial resolution of 330 nm, which was not shown before by basic confocal or ISM-based systems. Contrary to the majority of techniques, SPiRI method exhibits a low lateral-to-axial FWHM aspect ratio, which means a considerable improvement in 3D fluorescence imaging of cellular structures. As a demonstration of SPiRI application in biomedical sciences, we present a 3D structure of bacterial chromosome with excellent precision.