Image scanning microscopy: a vectorial physical optics analysis

Image scanning microscopy (ISM) achieves resolution beyond the diffraction limit by a factor of √2. However, prior ISM research predominantly employs scalar diffraction theory, neglecting critical physical effects such as polarization, aberrations, and Stokes shift. This paper presents a comprehensive vectorial ISM point spread function (PSF)...

Quantifying the minimum localization uncertainty of image scanning localization microscopy

Modulation enhanced single-molecule localization microscopy (meSMLM), where emitters are sparsely activated with sequentially applied patterned illumination, increases the localization precision over single-molecule localization microscopy (SMLM). The precision improvement of modulation enhanced SMLM is derived from retrieving the position of an...

Low-cost fluorescence microscope with microfluidic device fabrication for optofluidic applications

Optofluidic devices have revolutionized the manipulation and transportation of fluid at smaller length scales ranging from micrometers to millimeters. We describe a dedicated optical setup for studying laser-induced cavitation inside a microchannel. In a typical experiment, we use a tightly focused laser beam to locally evaporate the solution...

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...

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...

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high...

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...

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...

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...