Better 3D Inspection with Structured Illumination Part I: Signal Formation and Precision

For quality control in the factory, 3D-metrology faces increasing demands for high precision and for more space-bandwidth-speed-product SBSP (number of 3D-points/sec). As a potential solution, we will discuss Structured-Illumination Microscopy (SIM). We distinguish optically smooth and rough surfaces and develop a theoretical model of the signal formation for both surface species. This model is exploited to investigate the physical limits of the precision and to give rules to optimize the sensor parameters for best precision or high speed. This knowledge can profitably be combined with fast scanning strategies, to maximize the SBSP, which will be discussed in paper part II.Comment: 7 pages, 5 figures, submitted to Applied Optics on April 17, 201

Computational localization microscopy with extended axial range

A new single-aperture 3D particle-localization and tracking technique is presented that demonstrates an increase in depth range by more than an order of magnitude without compromising optical resolution and throughput. We exploit the extended depth range and depth-dependent translation of an Airy-beam PSF for 3D localization over an extended volume in a single snapshot. The technique is applicable to all bright-field and fluorescence modalities for particle localization and tracking, ranging from super-resolution microscopy through to the tracking of fluorescent beads and endogenous particles within cells. We demonstrate and validate its application to real-time 3D velocity imaging of fluid flow in capillaries using fluorescent tracer beads. An axial localization precision of 50 nm was obtained over a depth range of 120μm using a 0.4NA, 20× microscope objective. We believe this to be the highest ratio of axial range-to-precision reported to date

High-resolution transport-of-intensity quantitative phase microscopy with annular illumination

For quantitative phase imaging (QPI) based on transport-of-intensity equation (TIE), partially coherent illumination provides speckle-free imaging, compatibility with brightfield microscopy, and transverse resolution beyond coherent diffraction limit. Unfortunately, in a conventional microscope with circular illumination aperture, partial coherence tends to diminish the phase contrast, exacerbating the inherent noise-to-resolution tradeoff in TIE imaging, resulting in strong low-frequency artifacts and compromised imaging resolution. Here, we demonstrate how these issues can be effectively addressed by replacing the conventional circular illumination aperture with an annular one. The matched annular illumination not only strongly boosts the phase contrast for low spatial frequencies, but significantly improves the practical imaging resolution to near the incoherent diffraction limit. By incorporating high-numerical aperture (NA) illumination as well as high-NA objective, it is shown, for the first time, that TIE phase imaging can achieve a transverse resolution up to 208 nm, corresponding to an effective NA of 2.66. Time-lapse imaging of in vitro Hela cells revealing cellular morphology and subcellular dynamics during cells mitosis and apoptosis is exemplified. Given its capability for high-resolution QPI as well as the compatibility with widely available brightfield microscopy hardware, the proposed approach is expected to be adopted by the wider biology and medicine community.Comment: This manuscript was originally submitted on 20 Feb. 201

A Study on The Use of Double Helix Point Spread Functions in 3D Fluorescence Microscopy

Noninvasive three-dimensional (3D) imaging of thick samples in fluorescence microscopy is traditionally achieved via the method of optical sectioning where multiple two-dimensional images of an object acquired while focusing at different depths within the object. Defocus, spherical aberrations (SA) and photo bleaching are known to affect the accuracy achieved in 3D images. Double-Helix point spread functions (DH-PSF), the result of PSF engineering, have been used for super localization of point sources in 3D samples. The unique DH-PSF design features 2 dominant lobes in the image plane which appear to rotate as the axial (z) position of a point light source with respect to the imaging lens is changed. Thus, the angular orientation of the DH-PSF lobes encodes the imaging depth. The DH-PSF lobes are horizontal when the emitter is in focus. As the emitter is moved toward the objective lens, the DH-PSF lobes rotate, however, if the emitter is moved away from the objective the lobes rotate in the opposite direction. In this thesis, the effect of the DH-PSF in 3D optical sectioning microscopy is studied for different imaging conditions using simulations that introduce SA and noise. The frequency domain features of the DH-PSF were studied and were shown to preserve the rotation characteristic that encodes depth information. In addition frequency analysis shows that the DH-PSFs are less sensitive to defocus and SA at small imaging depths than the conventional PSF. The effect of SA is shown to cause an error in the super localization of emitters which it was successfully corrected by a new methodology developed in this thesis. Different approaches to estimate intensity and location of structures from computer generated images were investigated. The objects were effectively recovered from the images with and without SA and noise. Thus from this study we conclude that DH-PSF based systems can be effectively integrated into the conventional fluorescent microscopy system to help in particle tracking, super localization and object ranging