Fluorescence imaging for whole slide scanning using LED-based color sequential illumination

In the field of pathology there is an ongoing transition to the use of Whole Slide Imaging (WSI) systems which scan tissue slides at intermediate resolution (0∼.25 μm) and high throughput (15mm²=min) to digital image files. Most scanners currently on the market are line-sensor based push-broom scanners for three-color (RGB)...

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...

Single emitter localization analysis in the presence of background

Localization microscopy for imaging at the nano-scale relies on the quality of fitting the emitter positions from the measured light spots. The type and magnitude of the noise in the detection process, the background light level and the Point Spread Function model that is used in the fit are of paramount importance for the precision and accuracy...

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...

Differences in Nuclear DNA Organization Between Lymphocytes, Hodgkin and Reed–Sternberg Cells Revealed by Structured Illumination Microscopy

Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first time, the organization of nuclear DNA and that...

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...

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...

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...

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...