Quantitative Analysis for Single-Molecule Localization Microscopy: “From PSF to Information”

Single-molecule localization microscopy (SMLM) revolutionized the field of microscopy by allowing imaging below the diffraction limit. For decades, the resolution of microscopy has been limited by Abbe’s diffraction limit [1]. This limit dictates that two emitters cannot be resolved when they are less than λ/2NA apart, with λ the emission wavelength of the emitter and NA the numerical aperture of the objective. For an average microscope operating in the visible light spectrum, this translates to an approximate distance of 200 nm. Single-molecule localization microscopy as described in Chapter 2 shared the 2014 Noble Prize in Chemistry for super-resolution microscopy together with stimulated emission depletion (STED) microscopy. SMLM overcomes the diffraction limit by employing the clever trick of letting emitters repeatedly switch on and off over time [2]. When imaging these blinking emitters for many frames, in each frame only a sparse subset of the fluorophores is emitting light, resulting in isolated diffraction-limited spots in the image. The location of every individual spot can be determined with nanometer precision by fitting a model of the point spread function (PSF). The final super-resolution image is a rendering of the coordinates of all calculated positions of the spots in all frames.