ABSTRACT
The localization precision of single fluorophores, as well as the resolution achieved with single-molecule localization microscopy (SMLM) or coordinate-targeted super-resolution methods, is ultimately limited by the photostability of fluorophores [1–3]. As described in Chapter 2, under ambient conditions, the typical lateral resolution lies in the range of 10–40 nm, whereas the axial resolution is usually 3–5 times worse. Hence, recently, efforts have been devoted to developing methods enabling fluorescence imaging at the 1–10 nm scale, which would provide true molecular resolution by surpassing the typical size of structural proteins and even reaching the size of the fluorophores. The photostability limitation can be bypassed by two approaches: through obtaining more fluorescence photons from a given position in the sample, or by extracting more information from the limited photon budget. Both avenues have been explored to achieve super-resolution imaging with sub-10 nm resolution. DNA-PAINT (DNA-Point Accumulation for Imaging in Nanoscale Topography) is a SMLM method that uses transient hybridization of fluorescently labeled single-stranded DNA sequences (imager strands) to the target molecules/positions of the sample, which are functionalized and mediated with complementary DNA sequences (docking strands). This enables constant inspection of the target molecule/position with multiple fluorophores with nearly unlimited fluorescent photons [4, 5]. Dynamic binding-unbinding of the imager and docking strands generates the necessary blinking for SMLM. The advantage of DNA-PAINT lies in the fact that the virtually unlimited localization events can be measured for each labeled position of the sample, which can lead to sub 10 nm resolution [6–8]. A second approach aims to extract more information about the molecular position from the limited photon budget using a sequence of spatially modulated excitation beams. The latter approach for the ultra-precise localization of single fluorophores was pioneered by the so-called MINFLUX concept [9–11] which uses beams comprising a minimum of light.
