Physics-driven universal twin-image removal network for digital in-line holographic microscopy.

Digital in-line holographic microscopy (DIHM) enables efficient and cost-effective computational quantitative phase imaging with a large field of view, making it valuable for studying cell motility, migration, and bio-microfluidics. However, the quality of DIHM reconstructions is compromised by twin-image noise, posing a significant challenge. Conventional methods for mitigating this noise involve complex hardware setups or time-consuming algorithms with often limited effectiveness. In this work, we propose UTIRnet, a deep learning solution for fast, robust, and universally applicable twin-image suppression, trained exclusively on numerically generated datasets. The availability of open-source UTIRnet codes facilitates its implementation in various DIHM systems without the need for extensive experimental training data. Notably, our network ensures the consistency of reconstruction results with input holograms, imparting a physics-based foundation and enhancing reliability compared to conventional deep learning approaches. Experimental verification was conducted among others on live neural glial cell culture migration sensing, which is crucial for neurodegenerative disease research.

Revisiting Javal's rule: a fresh and improved power vector approach according to age.

PURPOSE: The scientific community has established Javal's rule as a model linking refractive (RA) and keratometric (KA) astigmatism since its appearance more than 100 years ago. The aim was to improve the accuracy of this relationship according to subject's age by applying the power vector analysis. Posterior corneal curvature has also been studied. METHODS: The IOLMaster 700 optical biometer was used to measure the corneal thickness and the radius of curvature of the anterior and posterior corneal surfaces. Refractive error was determined by a non-cycloplegic subjective refraction process with trial lenses. Linear regression analyses were applied using J0 and J45 power vector components. An evaluation was carried out according to the subject's age resulting into eight regression relationships for each astigmatic vector component for each relationship. RESULTS: A total of 2254 right eyes from 2254 healthy subjects were evaluated. A trend towards against-the-rule astigmatism (ATR) was found with aging, both for refractive astigmatism (RA) and keratometric astigmatism (KA), with 95.2% of subjects under 20 years old having with-the-rule (WTR) KA, and only 22.8% above 79 years old. The following regression equations were found between RA and KA: [Formula: see text] = 0.73 × [Formula: see text] - 0.18 (R = 0.78) and [Formula: see text] = 0.70 × [Formula: see text] + 0.04 (R = 0.69) and between RA and total corneal astigmatism (TCA): [Formula: see text] = 0.73 × [Formula: see text] + 0.13 (R=0.78) and [Formula: see text] = 0.70 × [Formula: see text] - 0.06 (R = 0.68) for the whole sample, but with sensible differences among age groups, both in the slope and in the intercept. CONCLUSION: Ignoring the age of the subject when using Javal's rule could lead to an error in the final cylinder calculation that would increase in high astigmatisms. Applying this new power vector approach based on subject's age could improve the accuracy of the astigmatism prediction.

Subjective refraction using power vectors by updating a conventional phoropter with a Stokes lens for continuous astigmatic power generation.

PURPOSE: To implement a pure power vector method for monocular subjective refraction using a regular phoropter with the only modification being the inclusion of a Stokes lens. The proposed methodology was tested with three different Stokes lenses, and the results were compared with conventional clinical refraction procedures. METHODS: Power vector subjective refraction was performed by attaching a Stokes lens to the Risley prism holder. Stokes lenses allow for pure astigmatic compensation in the form of the J0 , J45 components while the spherical lenses in the phoropter allow determination of the spherical component in the form of M (spherical equivalent). The proposed routine is presented step-by-step using three Stokes lenses having different astigmatic powers. RESULTS: Monocular subjective refraction was performed on 26 healthy subjects with a mean age of 44 ± 16 years, mean spherical equivalent of -0.56 D (range -5.50 to +2.38 D) and refractive astigmatism ≤1.50 D. No differences were found between the results obtained with the conventional technique versus the vector-based procedure for the spherical equivalent (p = 0.28) or astigmatic components (p = 0.34). In addition, visual acuity (VA) was equivalent through the refractions measured with the conventional and vector procedures (p = 0.12). Repeatability coefficients for J0 and J45 with the new vector methodology were <0.38 D. CONCLUSIONS: The proposed routine could be helpful for cases where it is difficult to get a valid starting point for conventional refraction (e.g., irregular corneas and media opacities), for testing facilities with limited resources/equipment and/or for motivated clinicians who wish to know about alternative methods of refractive error determination.

Single capture bright field and off-axis digital holographic microscopy: publisher's note.

This publisher's note contains corrections to Opt. Lett.48, 876 (2023)10.1364/OL.478674.

Vecto-keratometry: determination of anterior corneal astigmatism in manual keratometers using power vectors.

BACKGROUND: A new keratometric routine that employs power vector management for manual keratometers is described. This study evaluates the agreement of the new proposed keratometric technique with the classical one. RESEARCH DESIGN AND METHODS: The applicability of a new keratometric routine was verified using Helmholtz's and Javal's keratometers. Results were obtained by two different and well-trained examiners over two different samples, one including 65 and the other 74 eyes, respectively. Both conventional keratometry and the newly proposed routine (named vecto-keratometry) were used in each eye to obtain the results. The clinical agreement between the methods was evaluated using Bland-Altman and Passing-Bablok analysis. RESULTS: For Helmholtz's keratometer, Bland-Altman plots showed good agreement between methods for both astigmatic components being J0 = 0.04 ± 0.20 D and J45 = -0.07 ± 0.17 D. For Javal's keratometer, Passing-Bablok regression test determined regression line for J0 difference as y0 = 1.03, confidence interval: [0.98, 1.10] and regression line for J45 difference as y45 = 0.97, confidence interval: [0.83, 1.12]. CONCLUSIONS: Vecto-keratometry provides accurate clinical results. It has been demonstrated that there are no significant differences between methods in any of the power vector astigmatic components; thus, both methods can be applied interchangeably.

Single-shot wavelength-multiplexed phase microscopy under Gabor regime in a regular microscope embodiment.

Phase imaging microscopy under Gabor regime has been recently reported as an extremely simple, low cost and compact way to update a standard bright-field microscope with coherent sensing capabilities. By inserting coherent illumination in the microscope embodiment and producing a small defocus distance of the sample at the input plane, the digital sensor records an in-line Gabor hologram of the target sample, which is then numerically post-processed to finally achieve the sample's quantitative phase information. However, the retrieved phase distribution is affected by the two well-known drawbacks when dealing with Gabor's regime, that is, coherent noise and twin image disturbances. Here, we present a single-shot technique based on wavelength multiplexing for mitigating these two effects. A multi-illumination laser source (including 3 diode lasers) illuminates the sample and a color digital sensor (conventional RGB color camera) is used to record the wavelength-multiplexed Gabor hologram in a single exposure. The technique is completed by presenting a novel algorithm based on a modified Gerchberg-Saxton kernel to finally retrieve an enhanced quantitative phase image of the sample, enhanced in terms of coherent noise removal and twin image minimization. Experimental validations are performed in a regular Olympus BX-60 upright microscope using a 20X 0.46NA objective lens and considering static (resolution test targets) and dynamic (living spermatozoa) phase samples.

Multi-Illumination Single-Holographic-Exposure Lensless Fresnel (MISHELF) Microscopy: Principles and Biomedical Applications.

Lensless holographic microscopy (LHM) comes out as a promising label-free technique since it supplies high-quality imaging and adaptive magnification in a lens-free, compact and cost-effective way. Compact sizes and reduced prices of LHMs make them a perfect instrument for point-of-care diagnosis and increase their usability in limited-resource laboratories, remote areas, and poor countries. LHM can provide excellent intensity and phase imaging when the twin image is removed. In that sense, multi-illumination single-holographic-exposure lensless Fresnel (MISHELF) microscopy appears as a single-shot and phase-retrieved imaging technique employing multiple illumination/detection channels and a fast-iterative phase-retrieval algorithm. In this contribution, we review MISHELF microscopy through the description of the principles, the analysis of the performance, the presentation of the microscope prototypes and the inclusion of the main biomedical applications reported so far.

Fluholoscopy-Compact and Simple Platform Combining Fluorescence and Holographic Microscopy.

The combination of different imaging modalities into single imaging platforms has a strong potential in biomedical sciences as it permits the analysis of complementary properties of the target sample. Here, we report on an extremely simple, cost-effective, and compact microscope platform for achieving simultaneous fluorescence and quantitative phase imaging modes with the capability of working in a single snapshot. It is based on the use of a single illumination wavelength to both excite the sample's fluorescence and provide coherent illumination for phase imaging. After passing the microscope layout, the two imaging paths are separated using a bandpass filter, and the two imaging modes are simultaneously obtained using two digital cameras. We first present calibration and analysis of both fluorescence and phase imaging modalities working independently and, later on, experimental validation for the proposed common-path dual-mode imaging platform considering static (resolution test targets, fluorescent micro-beads, and water-suspended lab-made cultures) as well as dynamic (flowing fluorescent beads, human sperm cells, and live specimens from lab-made cultures) samples.

Single capture bright field and off-axis digital holographic microscopy.

We report on a single capture approach for simultaneous incoherent bright field (BF) and laser-based quantitative phase imaging (QPI). Common-path digital holographic microscopy (DHM) is implemented in parallel with BF imaging within the optical path of a commercial optical microscope to achieve spatially multiplexed recording of white light images and digital off-axis holograms, which are subsequently numerically demultiplexed. The performance of the proposed multimodal concept is firstly determined by investigations on microspheres. Then, the application for label-free dual-mode QPI and BF imaging of living pancreatic tumor cells is demonstrated.

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.

Shadowfocimetry: adapting the holographic principle to a manual focimeter for visualization/marking of permanent engravings in progressive addition lenses.

Focimeters, especially manual versions, are the most used ophthalmic devices for dioptric power measurement in optometric clinical care. In the particular case of progressive addition lenses (PALs), they are used to determine far/near vision correction powers, but the user/clinician needs to know at which part of the PAL the measurement must be taken. For this reason, PALs have permanent engravings acting as reference marks to define the far/near vision areas for every PAL design. However, for several reasons these engravings are often difficult to localize and identify, making an accurate dioptric power determination difficult. In this Letter, we present an adaptation of the Gabor holographic principle to a manual focimeter and describe the methodology for the correct localization, visualization, and marking process of the reference engravings in PALs. Experimental results considering different types of PALs are included and the main limitations of the technique are also discussed.

Astigmatic Stokes lens revisited.

Stokes lenses are variable power astigmatic lenses comprising of, in its standard version, two pure cylindrical lenses of equal but contrary power that rotate in opposite directions. Here, we present an optical device with variable and continuous astigmatic power which is based on a modified Stokes lens where two sphero-cylindrical lenses (in the form of pure astigmatic lenses) are combined in the classical way but merged with another fixed pure astigmatic lens for improving the capabilities of the resulting optical device concerning the expansion of the astigmatic range without worsening the dioptric power step resolution. The performance of this device is theoretically analyzed in virtue of the power vectors formalism including a three-dimensional (3-D) representation of the generated dioptric power as a function of both the meridian and the rotation angle between the cylinder's axes. In addition, we have assembled a lab-made prototype of the proposed modified Stokes lens and validated its theoretical behavior by dioptric power measurements with an automatic focimeter. As conventional Stokes lenses, the applications of this new optical device range from astigmatism compensation in optical instruments to measurement of refractive error in subjective routines with the previously commented improved capabilities.

Design, Calibration, and Application of a Robust, Cost-Effective, and High-Resolution Lensless Holographic Microscope.

Lensless holographic microscope (LHM) is an emerging very promising technology that provides high-quality imaging and analysis of biological samples without utilizing any lens for imaging. Due to its small size and reduced price, LHM can be a very useful tool for the point-of-care diagnosis of diseases, sperm assessment, or microfluidics, among others, not only employed in advanced laboratories but also in poor and/or remote areas. Recently, several LHMs have been reported in the literature. However, complete characterization of their optical parameters remains not much presented yet. Hence, we present a complete analysis of the performance of a compact, reduced cost, and high-resolution LHM. In particular, optical parameters such as lateral and axial resolutions, lateral magnification, and field of view are discussed into detail, comparing the experimental results with the expected theoretical values for different layout configurations. We use high-resolution amplitude and phase test targets and several microbeads to characterize the proposed microscope. This characterization is used to define a balanced and matched setup showing a good compromise between the involved parameters. Finally, such a microscope is utilized for visualization of static, as well as dynamic biosamples.

Hilbert phase microscopy based on pseudo thermal illumination in the Linnik configuration.

Quantitative phase microscopy (QPM) is often based on recording an object-reference interference pattern and its further phase demodulation. We propose pseudo Hilbert phase microscopy (PHPM) where we combine pseudo thermal light source illumination and Hilbert spiral transform (HST) phase demodulation to achieve hybrid hardware-software-driven noise robustness and an increase in resolution of single-shot coherent QPM. Those advantageous features stem from physically altering the laser spatial coherence and numerically restoring spectrally overlapped object spatial frequencies. The capabilities of PHPM are demonstrated by analyzing calibrated phase targets and live HeLa cells in comparison with laser illumination and phase demodulation via temporal phase shifting (TPS) and Fourier transform (FT) techniques. The performed studies verified the unique ability of PHPM to combine single-shot imaging, noise minimization, and preservation of phase details.

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.

Optical module for single-shot quantitative phase imaging based on the transport of intensity equation with field of view multiplexing.

We present a cost-effective, simple, and robust method that enables single-shot quantitative phase imaging (QPI) based on the transport of intensity equation (TIE) using an add-on optical module that can be assembled into the exit port of any regular microscope. The module integrates a beamsplitter (BS) cube (placed in a non-conventional way) for duplicating the output image onto the digital sensor (field of view - FOV - multiplexing), a Stokes lens (SL) for astigmatism compensation (introduced by the BS cube), and an optical quality glass plate over one of the FOV halves for defocusing generation (needed for single-shot TIE algorithm). Altogether, the system provides two laterally separated intensity images that are simultaneously recorded and slightly defocused one to each other, thus enabling accurate QPI by conventional TIE-based algorithms in a single snapshot. The proposed optical module is first calibrated for defining the configuration providing best QPI performance and, second, experimentally validated by using different phase samples (static and dynamic ones). The proposed configuration might be integrated in a compact three-dimensional (3D) printed module and coupled to any conventional microscope for QPI of dynamic transparent samples.

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.

Characterization of a compact low-cost Stokes lens for astigmatism compensation in optical instruments.

Variable power cross-cylinder lenses (or Stokes lenses) have been widely known in the literature for decades. In this paper, we describe how to build a low-cost Stokes lens and discuss its calibration and its application to two significant cases. The construction is in virtue of a phoropter's Risley prism mount for assembling a couple of equal but opposite sign cylindrical lenses (we have selected $\,\pm 1.50$±1.50 D). Thus, variable astigmatic power is achieved by relative rotation of the lenses in opposite directions, and the resulting astigmatic axis is defined by the global rotation of the device. Calibration measurements are performed using an automatic lensmeter (Topcon CL-300) and an aberrometer (Zeiss iProfiler plus) for low and high order, respectively, aberration characterization. The proposed device has been adapted to a manual Topcon LM-8 lensmeter and to a regular Olympus BX-60 upright microscope for experimental validation concerning astigmatism compensation in a digital microscope and astigmatism cancellation in ophthalmic lenses, respectively. The device can be easily adapted to any ophthalmic/optic instrument for the compensation and/or measurement of astigmatism up to a maximum range of $|3|$|3| D of astigmatism.

Automatic fringe pattern enhancement using truly adaptive period-guided bidimensional empirical mode decomposition.

Fringe patterns encode the information about the result of a measurement performed via widely used optical full-field testing methods, e.g., interferometry, digital holographic microscopy, moiré techniques, structured illumination etc. Affected by the optical setup, changing environment and the sample itself fringe patterns are often corrupted with substantial noise, strong and uneven background illumination and exhibit low contrast. Fringe pattern enhancement, i.e., noise minimization and background term removal, at the pre-processing stage prior to the phase map calculation (for the measurement result decoding) is therefore essential to minimize the jeopardizing effect the mentioned error sources have on the optical measurement outcome. In this contribution we propose an automatic, robust and highly effective fringe pattern enhancement method based on the novel period-guided bidimensional empirical mode decomposition algorithm (PG-BEMD). The spatial distribution of the fringe period is estimated using the novel windowed approach and then serves as an indicator for the truly adaptive decomposition with the filter size locally adjusted to the fringe pattern density. In this way the fringe term is successfully extracted in a single (first) decomposition component alleviating the cumbersome mode mixing phenomenon and greatly simplifying the automatic signal reconstruction. Hence, the fringe term is dissected without the need for modes selection nor summation. The noise removal robustness is ensured employing the block matching 3D filtering of the fringe pattern prior to its decomposition. Performance validation against previously reported modified empirical mode decomposition techniques is provided using numerical simulations and experimental data verifying the versatility and effectiveness of the proposed approach.