Photon efficient orientation estimation using polarization modulation in single-molecule localization microscopy

Combining orientation estimation with localization microscopy opens up the possibility to analyze the underlying orientation of biomolecules on the nanometer scale. Inspired by the recent improvement of the localization precision by shifting excitation patterns (MINFLUX, SIMFLUX), we have adapted the idea towards the modulation of excitation...

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

<i>Localization microscopy has circumvented the diffraction limit by sequentially</i> 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...

Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

Estimating the orientation and 3D position of rotationally constrained emitters with localization microscopy typically requires polarization splitting or a large engineered Point Spread Function (PSF). Here we utilize a compact modified PSF for single molecule emitter imaging to estimate simultaneously the 3D position, dipole orientation, and...

Waveguide-based total internal reflection fluorescence microscope enabling cellular imaging under cryogenic conditions

Total internal reflection fluorescence (TIRF) microscopy is an important imaging tool for the investigation of biological structures, especially the study on cellular events near the plasma membrane. Imaging at cryogenic temperatures not only enables observing structures in a near-native and fixed state but also suppresses irreversible photo...

Polarized stimulated-emission depletion and dark-state lifetime at vacuum and cryogenic temperature conditions

With the growing popularity of cryogenic correlative light and electron microscopy, it is becoming increasingly important to bridge the resolution gap between these two modalities. At cryogenic temperatures, the photon yield of fluorophores is a few orders of magnitude higher than at room temperature, enabling localization precisions on the...

High precision wavefront control in point spread function engineering for single emitter localization

Point spread function (PSF) engineering is used in single emitter localization to measure the emitter position in 3D and possibly other parameters such as the emission color or dipole orientation as well. Advanced PSF models such as spline fits to experimental PSFs or the vectorial PSF model can be used in the corresponding localization...

Photon count estimation in single-molecule localization microscopy

Recently, Franke, Sauer and van de Linde introduced a way to estimate the axial position of single-molecules (TRABI). To this end, they compared the detected photon count from a temporal radial-aperture-based intensity estimation to the estimated count from Gaussian point-spread function (PSF) fitting to the data. Empirically they found this...

Fluorescence Polarization Control for On-Off Switching of Single Molecules at Cryogenic Temperatures

Light microscopy, allowing sub-diffraction-limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the...

Single‐Molecule Switching: Fluorescence Polarization Control for On–Off Switching of Single Molecules at Cryogenic Temperatures

In article number 1700323, a sparsity‐inducing scheme based on widefield stimulated emission depletion, which requires stringent control of the polarization state of both the excitation and depletion laser, is introduced by Bernd Rieger and co‐workers for superresolution fluorescence microscopy. The ideal state involves excitation and depletion...

Impact of optical aberrations on axial position determination by photometry