Accurate automatic object 4D tracking in digital in-line holographic microscopy based on computationally rendered dark fields.

Building on Gabor seminal principle, digital in-line holographic microscopy provides efficient means for space-time investigations of large volumes of interest. Thus, it has a pivotal impact on particle tracking that is crucial in advancing various branches of science and technology, e.g., microfluidics and biophysical processes examination (cell motility, migration, interplay etc.). Well-established algorithms often rely on heavily regularized inverse problem modelling and encounter limitations in terms of tracking accuracy, hologram signal-to-noise ratio, accessible object volume, particle concentration and computational burden. This work demonstrates the DarkTrack algorithm-a new approach to versatile, fast, precise, and robust 4D holographic tracking based on deterministic computationally rendered high-contrast dark fields. Its unique capabilities are quantitatively corroborated employing a novel numerical engine for simulating Gabor holographic recording of time-variant volumes filled with predefined dynamic particles. Our solution accounts for multiple scattering and thus it is poised to secure an important gap in holographic particle tracking technology and allow for ground-truth-driven benchmarking and quantitative assessment of tracking algorithms. Proof-of-concept experimental evaluation of DarkTrack is presented via analyzing live spermatozoa. Software supporting both novel numerical holographic engine and DarkTrack algorithm is made open access, which opens new possibilities and sets the stage for democratization of robust holographic 4D particle examination.

Versatile optimization-based speed-up method for autofocusing in digital holographic microscopy.

We propose a speed-up method for the in-focus plane detection in digital holographic microscopy that can be applied to a broad class of autofocusing algorithms that involve repetitive propagation of an object wave to various axial locations to decide the in-focus position. The classical autofocusing algorithms apply a uniform search strategy, i.e., they probe multiple, uniformly distributed axial locations, which leads to heavy computational overhead. Our method substantially reduces the computational load, without sacrificing the accuracy, by skillfully selecting the next location to investigate, which results in a decreased total number of probed propagation distances. This is achieved by applying the golden selection search with parabolic interpolation, which is the gold standard for tackling single-variable optimization problems. The proposed approach is successfully applied to three diverse autofocusing cases, providing up to 136-fold speed-up.

Common-path intrinsically achromatic optical diffraction tomography.

In this work we propose an open-top like common-path intrinsically achromatic optical diffraction tomography system. It operates as a total-shear interferometer and employs Ronchi-type amplitude diffraction grating, positioned in between the camera and the tube lens without an additional 4f system, generating three-beam interferograms with achromatic second harmonic. Such configuration makes the proposed system low cost, compact and immune to vibrations. We present the results of the measurements of 3D-printed cell phantom using laser diode (coherent) and superluminescent diode (partially coherent) light sources. Broadband light sources can be naturally employed without the need for any cumbersome compensation because of the intrinsic achromaticity of the interferometric recording (holograms generated by -1st and +1st conjugated diffraction orders are not affected by the illumination wavelength). The results show that the decreased coherence offers much reduced coherent noise and higher fidelity tomographic reconstruction especially when applied nonnegativity constraint regularization procedure.

The Origin of the Cyathea delgadii Sternb. Somatic Embryos Is Determined by the Developmental State of Donor Tissue and Mutual Balance of Selected Metabolites.

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

FPM app: an open-source MATLAB application for simple and intuitive Fourier ptychographic reconstruction.

SUMMARY: Fourier ptychographic microscopy (FPM) is a computational microscopy technique that enables large field of view and high-resolution microscopic imaging of biological samples. However, the FPM does not yet have an adequately capable open-source software. In order to fill this gap we are presenting novel, simple, universal, semi-automatic and highly intuitive graphical user interface (GUI) open-source application called the FPM app enabling wide-scale robust FPM reconstruction. Apart from implementing the FPM in accessible GUI app, we also made several improvements in the FPM image reconstruction process itself, making the FPM more automatic, noise-robust and faster. AVAILABILITY AND IMPLEMENTATION: FPM app was implemented in MATLAB and all MATLAB codes along with standalone executable version of the FPM app and the online documentation are freely accessible at https://github.com/MRogalski96/FPM-app. Our exemplary FPM datasets may be downloaded at https://bit.ly/2MxNpGb. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Author Correction: Variational Hilbert Quantitative Phase Imaging.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Variational Hilbert Quantitative Phase Imaging.

Utilizing the refractive index as the endogenous contrast agent to noninvasively study transparent cells is a working principle of emerging quantitative phase imaging (QPI). In this contribution, we propose the Variational Hilbert Quantitative Phase Imaging (VHQPI)-end-to-end purely computational add-on module able to improve performance of a QPI-unit without hardware modifications. The VHQPI, deploying unique merger of tailored variational image decomposition and enhanced Hilbert spiral transform, adaptively provides high quality map of sample-induced phase delay, accepting particularly wide range of input single-shot interferograms (from off-axis to quasi on-axis configurations). It especially promotes high space-bandwidth-product QPI configurations alleviating the spectral overlapping problem. The VHQPI is tailored to deal with cumbersome interference patterns related to detailed locally varying biological objects with possibly high dynamic range of phase and relatively low carrier. In post-processing, the slowly varying phase-term associated with the instrumental optical aberrations is eliminated upon variational analysis to further boost the phase-imaging capabilities. The VHQPI is thoroughly studied employing numerical simulations and successfully validated using static and dynamic cells phase-analysis. It compares favorably with other single-shot phase reconstruction techniques based on the Fourier and Hilbert-Huang transforms, both in terms of visual inspection and quantitative evaluation, potentially opening up new possibilities in QPI.

Grating deployed total-shear 3-beam interference microscopy with reduced temporal coherence.

Interference microscopy is a powerful optical imaging technique providing quantitative phase distribution information to characterize various type technical and biomedical objects. Static and dynamic objects and processes can be investigated. In this paper we propose very compact, common-path and partially coherent diffraction grating-based interference microscopy system for studying small objects like single cells with low densities being sparsely distributed in the field of view. Simple binary amplitude diffraction grating is the only additional element to be introduced into a conventional microscope optical system. By placing it at a proper distance in front of the microscope image plane the total-shear operation mode is deployed resulting in interferograms of the object-reference beam type. Depending on the grating to image plane separation distance two or three-beam interferograms are generated. The latter ones are advantageous since they contain achromatic second harmonics in the interferogram intensity distributions. This feature enables to use reduced temporal coherence light sources for the microscope to reduce coherent noise and parasitic interference patterns. For this purpose we employ the laser diode with driving current below the threshold one. Results of conducted experiments including automatic computer processing of interferograms fully corroborate analytical description of the proposed method and illustrate its capabilities for studying static and dynamic phase objects.

Numerically Enhanced Stimulated Emission Depletion Microscopy with Adaptive Optics for Deep-Tissue Super-Resolved Imaging.

In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilizing a high-power depletion laser (increasing the risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here, we demonstrate a custom-built STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating tissue. We introduce and investigate a state of the art image denoising method by block-matching and collaborative 3D filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise and further enhance the image quality. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well established Fourier ring correlation metric. Results achieved through the combination of aberration correction and tailored image processing are experimentally validated through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under an 80 µm thick layer of tissue with lateral and axial resolution of 204 and 310 nm, respectively.

Single-shot two-frame π-shifted spatially multiplexed interference phase microscopy.

Single-shot, two-frame, π-shifted spatially multiplexed interference microscopy (π-SMIM) is presented as an improvement to previous SMIM implementations, introducing a versatile, robust, fast, and accurate method for cumbersome, noisy, and low-contrast phase object analysis. The proposed π-SMIM equips a commercially available nonholographic microscope with a high-speed (video frame rate) enhanced quantitative phase imaging (QPI) capability by properly placing a beam-splitter in the microscope embodiment to simultaneously (in a single shot) record two holograms mutually phase shifted by π radians at the expense of reducing the field of view. Upon subsequent subtractive superimposition of holograms, a π-hologram is generated with reduced background and improved modulation of interference fringes. These features determine superior phase retrieval quality, obtained by employing the Hilbert spiral transform on the π-hologram, as compared with a single low-quality (low signal-to-noise ratio) hologram analysis. In addition, π-SMIM enables accurate in-vivo analysis of high dynamic range phase objects, otherwise measurable only in static regime using time-consuming phase-shifting. The technique has been validated utilizing a 20 × / 0.46 NA objective in a regular Olympus BX-60 upright microscope for QPI of different lines of prostate cancer cells and flowing microbeads.

Full volume super-resolution imaging of thick mitotic spindle using 3D AO STED microscope.

Stimulated emission depletion (STED) nanoscopy is one of a suite of modern optical microscopy techniques capable of bypassing the conventional diffraction limit in fluorescent imaging. STED makes use of a spiral phase mask to enable 2D super-resolution imaging whereas to achieve full volumetric 3D super-resolution an additional bottle-beam phase mask must be applied. The resolution achieved in biological samples 10 µm or thicker is limited by aberrations induced mainly by scattering due to refractive index heterogeneity in the sample. These aberrations impact the fidelity of both types of phase mask, and have limited the application of STED to thicker biological systems. Here we apply an automated adaptive optics solution to correct the performance of both STED masks, enhancing robustness and expanding the capabilities of this nanoscopic technique. Corroboration in terms of successful high-quality imaging of the full volume of a 15µm mitotic spindle with resolution of 50nm x 50nm x 150nm achieved in all three dimensions is presented.

Three-level transmittance 2D grating with reduced spectrum and its self-imaging.

A simple method for generating 2D binary amplitude structure with additive superimposition of mutually orthogonal 1D amplitude gratings is proposed. Its implementation requires software generated three binary amplitude gratings, i.e., the crossed Ronchi, checker board and 1D Ronchi gratings with aspect ratio equal to 0.5. Their computer processing involves only two steps. First the checker grating is multiplied by a high frequency 1D grating. Next the product is added to the crossed grating. In result 3-level transmittance (0, 0.5, 1) hybrid diffraction structure is obtained. The intermediate level results from the use of a dense 1D grating. The zero diffraction order, well separated from the rest of the spectrum, consists of crossed spectra of additively superimposed 1D Ronchi gratings. Detailed heuristic explanation of the process aided by spectrum domain analyses is presented. Additionally, simulations and experiments conducted in the Fresnel diffraction field exemplify the invented structure properties in comparison with the multiplicative superimposition crossed Ronchi grating. Up to authors' best knowledge the Fresnel field (self-imaging phenomenon or Talbot effect) properties of 2D periodic structure with additive superimposition of component 1D gratings have not been published in the literature.