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...

High-speed multicolor structured illumination microscopy using a hexagonal single mode fiber array

Structured illumination microscopy (SIM) is a widely used imaging technique that doubles the effective resolution of widefield microscopes. Most current implementations rely on diffractive elements, either gratings or programmable devices, to generate structured light patterns in the sample. These can be limited by spectral efficiency, speed, or...

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...

Detecting structural heterogeneity in single-molecule localization microscopy data

Particle fusion for single molecule localization microscopy improves signal-to-noise ratio and overcomes underlabeling, but ignores structural heterogeneity or conformational variability. We present a-priori knowledge-free unsupervised classification of structurally different particles employing the Bhattacharya cost function as dissimilarity...

3D particle averaging and detection of macromolecular symmetry in localization microscopy

Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D...

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...

Automated detection of cribriform growth patterns in prostate histology images

Cribriform growth patterns in prostate carcinoma are associated with poor prognosis. We aimed to introduce a deep learning method to detect such patterns automatically. To do so, convolutional neural network was trained to detect cribriform growth patterns on 128 prostate needle biopsies. Ensemble learning taking into account other tumor...

Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy

Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of...

Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or...

Three-dimensional structured illumination microscopy using Lukosz bound apodization reduces pixel negativity at no resolution cost

The quality of the reconstructed image in structured illumination microscopy (SIM) depends on various aspects of the image filtering process. To optimize the trade-off between resolution and ringing artifacts, which lead to negative intensities, we extend Lukosz-bound filtering to 3D SIM and derive the parametrization of the 3D SIM cut-off. We...

Image filtering in structured illumination microscopy using the Lukosz bound

Various aspects of image filtering affect the final image quality in Structured Illumination Microscopy, in particular the regularization parameter and type of regularization function, the relative height of the side bands, and the shape of the apodization function. We propose an apodization filter without adjustable parameters based on the...

High NA diffractive array illuminators and application in a multi-spot scanning microscope

Array illuminators generating spots with high NA at high efficiency are presented. They are designed via application of high-NA scalar optics methods, and implemented as periodic binary phase structures. These array illuminators are used in a multi-spot scanning microscope for scanning large sample areas with a relatively high resolution.

Position and orientation estimation of fixed dipole emitters using an effective Hermite point spread function model

We introduce a method for determining the position and orientation of fixed dipole emitters based on a combination of polarimetry and spot shape detection. A key element is an effective Point Spread Function model based on Hermite functions. The model offers a good description of the shape variations with dipole orientation and polarization...

Optical trapping at low numerical aperture

A theory of optical trapping at low Numerical Aperture (NA) is presented. The theory offers an analytical description of the competition between the stabilizing gradient and destabilizing scattering force. The trade-off can be characterized by a single dimensionless trapping parameter, which increases with bead size to wavelength ratio ?/?, m,...

A fast algorithm for computing and correcting the CTF for tilted, thick specimens in TEM

Today, the resolution in phase-contrast cryo-electron tomography is for a significant part limited by the contrast transfer function (CTF) of the microscope. The CTF is a function of defocus and thus varies spatially as a result of the tilting of the specimen and the finite specimen thickness. Models that include spatial dependencies have not...