Figure 3.

Three-dimensional (3D) super-resolution fluorescence imaging. (a) To extract the location of individual single-molecule emitters with high precision in all three spatial dimensions, the symmetry of the standard microscope PSF (i) must be broken and the PSF re-designed to encode information in the z (axial) direction by a well-defined shape change. Widely adopted schemes include: (ii) an astigmatic PSF, where the slowly changing ellipticity of the single-molecule image can be calibrated to provide a z estimate. (iii) the double-helix (DH) PSF features two well-defined spots revolving around a common center as a function of z. (iv) cork screw PSF (one revolving spot), (v) bi-plane methods assess the relative detected brightness of images formed in two shifted image planes. (vi) 4Pi axial localization methods rely on interferometric detection and two matched objective lenses to collect fluorescence from both sides of the sample. (b) Microtubules in mammalian cells extending over a large axial range can be readily imaged with the DH-PSF. (c) Visualization of the cytoskeletal protein filament of CreS in pre-divisional C. crescentus bacterial cells, jointly with the cell surface by the PAINT approach. (d) Quantitative co-localization of a further protein, PopZ (shown in red). The number density of PopZ in the polar nano-domains was demonstrated to be constant between cells. Both (c) and (d) recorded by DH-PSF microscopy. (e) Cell-wide arrangement of F-actin by dual-objective astigmatic imaging. Examples reproduced with permission from [32] (b), [88] (c), [89] (d), [33] (e).