Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence

Abstract

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound

“…In order to be able to determine the defocus, we considered an astigmatic imaging approach, which allows one to obtain x, y, and d for the dipole emitter simultaneously. Such an approach is routine and available in many laboratories using single-molecule microscopy [18][19][20]. We assumed here a very weak astigmatism corresponding to a shift of the two foci of approximately 1.4 µm.…”

“…1b). Astigmatism-based superresolution microscopy is widely used for estimating the z-position of isotropic PSFs [18][19][20]. In order to determine the dipole orientation, two categories of approaches can be distinguished: (i) In spot shape analysis, the delicate way in which the dipole orientation of a fluorophore affects the shape of the PSF (e.g.…”

Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy

“…In order to be able to determine the defocus, we considered an astigmatic imaging approach, which allows one to obtain x, y, and d for the dipole emitter simultaneously. Such an approach is routine and available in many laboratories using single-molecule microscopy [18][19][20]. We assumed here a very weak astigmatism corresponding to a shift of the two foci of approximately 1.4 μm.…”

“…A well-established method which allows for defocus estimation is to introduce a weak cylindrical lens in the optical path, resulting in an elliptical distortion of the PSF of an emitter above or below the focal plane [17] (compare Fig 1b). Astigmatism-based superresolution microscopy is widely used for estimating the z-position of isotropic PSFs [18][19][20]. In order to determine the dipole orientation, two categories of approaches can be distinguished: (i) In spot shape analysis, the delicate way in which the dipole orientation of a fluorophore affects the shape of the PSF (e.g.…”