ZIMFLUX: Single molecule localization microscopy with patterned illumination in 3D

Three dimensional modulation-enhanced single-molecule localization techniques, such as ModLoc, offer advancements in axial localization precision across the entire field of view and axial capture range, by applying phase shifting to the illumination pattern. However, this improvement is limited by the pitch of the illumination pattern that...

Bayesian posterior density estimation reveals degeneracy in three-dimensional multiple emitter localization

Single-molecule localization microscopy requires sparse activation of emitters to circumvent the diffraction limit. In densely labeled or thick samples, overlap of emitter images is inevitable. Single-molecule localization of these samples results in a biased parameter estimate with a wrong model of the number of emitters. On the other hand,...

Three-dimensional localization microscopy with increased axial precision through TIRF angle modulation

To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial resolution of microscopes however, is a limiting factor in fluorescence imaging, correspondingly in fluorescence based single molecule localization microscopy (SMLM). Here, we present...

SOLEIL: Single-objective lens inclined light sheet localization microscopy

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the...

Precision in iterative modulation enhanced single-molecule localization microscopy

Modulation enhanced single-molecule localization microscopy (meSMLM) methods improve the localization precision by using patterned illumination to encode additional position information. Iterative meSMLM (imeSMLM) methods iteratively generate prior information on emitter positions, used to locally improve the localization precision during...

Global correction of optical distortions in multicolor single-molecule microscopy using Zernike polynomial gradients

Accurate image alignment is critical in multicolor single-molecule fluorescence microscopy. Global alignment using affine transformations leaves residual errors due to the nonlinearity of the distortions, which decreases the effective field of view. Subsequent local refinement demands either large amounts of reference data and processing time...

Single objective tilted lightsheet for three-dimensional localization microscopy

Optical sectioning technologies achieve high precision localization by reducing the background photon count. We use tilted light-sheet microscopy to achieve optical sectioning in localization microscopy, enabling thick sample observation and low background photon count images. A deformable mirror was incorporated to generate a tetrapod point...

Drift correction in localization microscopy using entropy minimization

Localization microscopy offers resolutions down to a single nanometer but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from the drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal...

Theoretical minimum uncertainty of single-molecule localizations using a single-photon avalanche diode array

Single-photon avalanche diode (SPAD) arrays can be used for single-molecule localization microscopy (SMLM) because of their high frame rate and lack of readout noise. SPAD arrays have a binary frame output, which means photon arrivals should be described as a binomial process rather than a Poissonian process. Consequentially, the theoretical...

Localization microscopy at doubled precision with patterned illumination

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold...