Slight Defocus Research Articles

Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy.

Single molecule localization microscopy (SMLM) has the potential to resolve structural details of biological samples at the nanometer length scale. Compared to room temperature experiments, SMLM performed under cryogenic temperature achieves higher photon yields and, hence, higher localization precision. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters with fixed orientation. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of 1 nm, thereby reaching the corresponding Cramér Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments.

Partially coherent beam smoothing using a microlens array

The main issue in applying a microlens array as a beam homogenizer is the high intensity contrast resulting from the periodic structure. We present a practical solution to eliminate speckle patterns by using a diffuser that can convert a coherent source into a Gaussian Schell source with an adjustable coherence length. Based on numerical simulation and experimental results, the microlens should have the Fresnel number above 15, a large pitch when the target plane is fixed, and an appropriate D/σf ratio to obtain a spot with high uniformity. Moreover, a slight defocus of the observation surface within the range of ±1mm has a negligible effect on the uniformity of shaped spots the homogenized spot. Finally, the applications of the findings are presented.

Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence.

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

Sharpening Image Details Using Local Phase Congruency Analysis

We suggest a method for sharpening an image or video stream without using convolution, as in unsharp masking, or deconvolution, as in constrained least-squares filtering. Instead, our technique is based on a local analysis of phase congruency and hence focuses on perceptually important details. The image is partitioned into overlapping tiles, and is processed tile by tile. We perform a Fourier transform for each of the tiles, and define congruency for each of the components in such a way that it is large when the component's neighbours are aligned with it, and small otherwise. We then amplify weak components with high phase congruency and reduce strong components with low phase congruency. Following this method, we avoid strengthening the Fourier components corresponding to sharp edges, while amplifying those details that underwent a slight or moderate defocus blur. The tiles are then seamlessly stitched. As a result, the image sharpness is improved wherever perceptually important details are present.

The influence of constant slight myopic defocus prescribed in the binocular and alternating monocular spectacle formats on the onset and progression of myopia in the children

Objective. To elucidate the long-term influence of constant slight myopic defocus prescribed in the binocular and the alternating monocular spectacle formats on the dynamics of refraction and its components in the children. Material and methods. The study involved 129 children (258 eyes) at the age varying from 5 to 12 years. The patients were divided into four groups. Group 1 was comprised of 48 children at the age of 5-8 years presenting with emmetropia and risk factors of the development of myopia. Group 2 consisted of 46 children at the age from 7 to 11 years with slight myopia between -0.75 and -2.25 D to whom the alternating continuous wearing of two pairs of spectacles was prescribed. The first control group was composed of 15 children (30 eyes) at the age from 6 to 9 (mean 7.5 ± 1.4) years presenting with pseudomyopia without correction. The second control group was comprised of 20 children (40 eyes) at the age from 7 to 12 (mean 9.7 ± 1.2) years having slight myopia and wearing conventional spectacle correction. Results. The children of group 1subjected to constant slight myopic defocus during one month experienced a shift of refraction toward hypermetropia attributable to the thinning of the lens and deepening of the anterior chamber. None of the children in this group developed myopia during the follow-up period of up to 9 years. In the patients of group 2, refraction remained unaltered during either 4 years (81.8% of the cases) or 7 years (66.6% of the cases). An insignificant increase in the length of the antero-posterior axis was documented in these children along with the significant increase of the horizontal diameter (HD) of the eyeball. The dynamic observation of the children of both control groups during 3 years has demonstrated the strengthening of cycloplegic refraction associated with the significant increase of the length of the antero-posterior axis and the insignificant increase of the transverse diameter of the eyeball. Conclusion. The permanent slight myopic defocusing of the image in the binocular spectacle format slows down the growth of the eyes and the shift of refraction toward myopia in the children with mild hypermetropia, emmetropia, and slight myopia. The proposed method for alternating monolateral constant slight myopic defocus inhibits progression of myopia in 81.8% of the children presenting with mild myopia during 4 years and in 66.6% of them during 7 years.

High-speed three-dimensional shape measurement for dynamic scenes using bi-frequency tripolar pulse-width-modulation fringe projection

This paper introduces a high-speed three-dimensional (3-D) shape measurement technique for dynamic scenes by using bi-frequency tripolar pulse-width-modulation (TPWM) fringe projection. Two wrapped phase maps with different wavelengths can be obtained simultaneously by our bi-frequency phase-shifting algorithm. Then the two phase maps are unwrapped using a simple look-up-table based number-theoretical approach. To guarantee the robustness of phase unwrapping as well as the high sinusoidality of projected patterns, TPWM technique is employed to generate ideal fringe patterns with slight defocus. We detailed our technique, including its principle, pattern design, and system setup. Several experiments on dynamic scenes were performed, verifying that our method can achieve a speed of 1250 frames per second for fast, dense, and accurate 3-D measurements.

Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy.

The cytoskeleton consists of complex arrays of fibers that play indispensable roles in cell structure and function. The cytoskeletal fibers are concertedly involved in numerous cellular processes, including cell adhesion, locomotion, intracellular transport, and cell division. The organization of the cytoskeleton was extensively studied, mainly by immunofluorescence microscopy, yet these studies were mostly qualitative, and a reliable quantitative approach for determining fiber structure and distribution is still missing. In this study we developed algorithms for filament feature extraction, based on fluorescence microscopy. These algorithms are robust against blurring by slight defocus, high background, and noise, and are applicable to both fixed, immunolabeled cells and live cells expressing fluorescently tagged cytoskeletal proteins. The implemented FiberScore program is used in order to recognize, segment, and quantify various structural parameters of the cytoskeleton, including total fiber-associated fluorescence, as well as fiber length and orientation. Furthermore, these parameters can be determined for different cytoskeletal proteins in the same sample tagged with multiple-fluorescent labels, and the results can be correlated with other cellular parameters. FiberScore was used here for the quantification of simultaneous changes in microtubule and actin filaments induced by the microtubule-disrupting drug nocodazole. Actin filaments, which are reported to respond reciprocally to microtubule disruption, are found to be affected by both immediate and delayed signals. Analysis of the organization of fibers by the FiberScore algorithm allows quantification of the cytoskeletal signature of cells and offers reliable multiparametric functional assays for effects of drugs and other perturbations evaluated on a cell-by-cell basis.

Segmented refraction of the crystalline lens as a prerequisite for the occurrence of monocular polyplopia, increased depth of focus, and contrast sensitivity function notches

Theoretical computations of modulation transfer functions (MTF’s) of the optical system of the human eye have shown that irregular aberration consisting of a small circular segment with refractive power slightly different from the surround introduces at higher spatial frequencies (>20 cpd) an enhancement of the retinal image contrast on flanks of the optimum-focus plane. When the pupil size is larger than 3 mm, enhancement is substantial; as a result, multiple foci appear at the affected, higher spatial frequencies and generate a greater depth of focus. The contrast enhancement also produces troughs on either flank of the optimum-focus plane. With slight coincident defocus (±0.5 diopter) of the retinal image of a sine-wave grating, notches in the MTF curves, with a contrast reduction in the intermediate frequency range of a factor of 2 to 3 and a low cutoff spatial frequency of ~3 cycles/deg, are produced. In our theoretical study, multiple foci, monocular polyplopia, and increased depth of focus are implicated in the generation of contrast sensitivity function (CSF) notches. It is demonstrated that CSF notches of optical origin can extend to lower spatial frequencies (<10 cycles/deg). As a result, before the presence of a CSF notch can be attributed to neurological abnormality, optical factors, including irregular aberrations, must be eliminated.