Development of a DMD-based fluorescence microscope

We present a versatile fluorescence microscope, built by complementing a conventional fluorescence microscope with a digital micro-mirror device (DMD) in the illumination path. Arbitrary patterns can be created on the DMD and projected onto the sample. This patterned illumination can be used to improve lateral and axial resolution over the...

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...

Simultaneous measurement of position and color of single fluorescent emitters using diffractive optics

We propose a method for simultaneously measuring the position and emission color of single fluorescent emitters based on the use of a large pitch diffraction grating in the emission light path. The grating produces satellite spots adjacent to the main spot; the relative distance between the spots is a measure for the emission wavelength. We...

Automated podosome identification and characterization in fluorescence microscopy images

Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures...

Precise and unbiased estimation of astigmatism and defocus in transmission electron microscopy

Defocus and twofold astigmatism are the key parameters governing the contrast transfer function (CTF) in transmission electron microscopy (TEM) of weak phase objects. We present a new algorithm to estimate these aberrations and the associated uncertainties. Tests show very good agreement between simulated and estimated defocus and astigmatism....

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...

Accuracy of the Gaussian Point Spread Function model in 2D localization microscopy

The Gaussian function is simple and easy to implement as Point Spread Function (PSF) model for fitting the position of fluorescent emitters in localization microscopy. Despite its attractiveness the appropriateness of the Gaussian is questionable as it is not based on the laws of optics. Here we study the effect of emission dipole orientation in...

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

Sputtering limits versus signal-to-noise limits in the observation of Sn balls in a Ga+ microscope

In principle, a scanning ion microscope can produce smaller probe sizes than a scanning electron microscope because the diffraction contribution is smaller. However, the imaging resolution is often severely limited by the sputtering damage. In this article, an experimental procedure to establish the limit of a focused ion beam system for imaging...

Biological applications of an LCoS-BASED PROGRAMMABLE ARRAY MICROSCOPE (PAM)

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS)...

A new optical method for characterizing single molecule interactions based on dark field microscopy

Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been...