Localization microscopy of constrained fluorescent molecules: Pushing towards Ångström-scale resolution through cryogenics

Localization microscopy has circumvented the diffraction limit by sequentially imaging individual light emitting molecules at a time. The position of these individual molecules can be determined and a super-resolution reconstruction is made with improved resolution. Normally freely rotating emitters are used such that the point spread function (PSF) is rotationally symmetric and only minor errors in the localization process are made by approximating the PSF with a Gaussian. The precision with which the individual emitters can be localized scales with the 1/√N, N the number of detected photons so that more detected photons leads to a better localization precision. However, the emission of fluorescent molecules is limited by photobleaching, a light induced chemical reaction to a permanent non-fluorescent state. In this thesis we investigate the effect of cooling the sample to cryogenic temperatures with liquid nitrogen. This reduces the chemical reaction rates and improves photostability more than 100 fold. To use localization microscopy it is necessary to switch the fluorescent molecules between an on-state and off-state, this turns out to be difficult at cryogenic temperatures. Standard methods used at room temperature in aqueous media do not work. As the molecules are frozen in place at cryogenic temperatures we use polarized light to selectively image molecules with certain orientations at a time. To realize this it is necessary to generate pure linear polarization with an arbitrary orientation in the sample plane. By calibrating the phase difference induced by the dichroic mirrors this can be achieved, effectively modulating the fluorescence of fixed dipole emitters at cryogenic temperatures. The addition of an orthogonal linearly polarized stimulated emission depletion (STED) beam narrows the orientational distribution of fluorescing molecules. This method does induce some degree of sparsity, however, it is not enough for localization microscopy of dense biological samples. Furthermore, the STED process reduces the photon yield of single molecules. This is presumably caused by the long dark-state recovery measured on fluorescent molecules in vacuum and at cryogenic temperatures. Localization microscopy of fixed or orientationally constrained emitters has long been avoided as the orientation of individual molecules leads to bias in the localizations. There are various ways to eliminate this bias but they reduce the amount of information that can be extracted from the sample. By fixing the orientation of fluorescent emitters to biomolecules of interest they become reporters for the orientation of the biomolecules. We have devised the so-called Vortex PSF with which the orientation, 3D position and degree of rotational constraint can be extracted from a single image. Alternatively the orientation of single-molecules can be probed with varying polarization states over multiple frames achieving a better precision with less photons.