Localization microscopy of single molecules enhanced by 3D imaging and light sheet illumination

Abstract

Single-molecule super-resolution fluorescence microscopy has produced a revolution in the optical resolution available for study of the nanoscale details of cells and materials. The early work on two-dimensional imaging yielded images of samples that spanned only a small axial range on the order of ~500 nm. Much more useful information can be obtained in complex extended samples using three-dimensional (3D) single-molecule localization. Without 3D capability, visualization of structures extending in the axial direction can easily be missed or confused and the understanding of the science compromised. A variety of methods for obtaining 3D super-resolution images have been devised, each with their own strengths and weaknesses, ranging from point-spread-function engineering to steep axial illumination gradients. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions presents additional challenges due to increased background and large volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage. Beyond these developments, there is a continuing need for better controllable fluorophores, additional novel optical system designs, and enhanced mathematical and statistical signal processing ideas to accurately extract the maximum information available from single emitters in the shortest possible time.