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.

Soft Contact Lens Engraving Characterization by Wavefront Holoscopy.

Permanent engravings on contact lenses provide information about the manufacturing process and lens positioning when they are placed on the eye. The inspection of their morphological characteristics is important, since they can affect the user's comfort and deposit adhesion. Therefore, an inverted wavefront holoscope (a lensless microscope based on Gabor's principle of in-line digital holography) is explored for the characterization of the permanent marks of soft contact lenses. The device, based on an in-line transmission configuration, uses a partially coherent laser source to illuminate the soft contact lens placed in a cuvette filled with a saline solution for lens preservation. Holograms were recorded on a digital sensor and reconstructed by back propagation to the image plane based on the angular spectrum method. In addition, a phase-retrieval algorithm was used to enhance the quality of the recovered images. The instrument was experimentally validated through a calibration process in terms of spatial resolution and thickness estimation, showing values that perfectly agree with those that were theoretically expected. Finally, phase maps of different engravings for three commercial soft contact lenses were successfully reconstructed, validating the inverted wavefront holoscope as a potential instrument for the characterization of the permanent marks of soft contact lenses. To improve the final image quality of reconstructions, the geometry of lenses should be considered to avoid induced aberration effects.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hilbert-Huang single-shot spatially multiplexed interferometric microscopy.

Hilbert-Huang single-shot spatially multiplexed interferometric microscopy (H2S2MIM) is presented as the implementation of a robust, fast, and accurate single-shot phase estimation algorithm with an extremely simple, low-cost, and highly stable way to convert a bright field microscope into a holographic one using partially coherent illumination. Altogether, H2S2MIM adds high-speed (video frame rate) quantitative phase imaging capability to a commercially available nonholographic microscope with improved phase reconstruction (coherence noise reduction). The technique has been validated using a 20×/0.46 NA objective in a regular Olympus BX-60 upright microscope for static, as well as dynamic, samples showing perfect agreement with the results retrieved from a temporal phase-shifting algorithm.

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

Single-shot, dual-mode, water-immersion microscopy platform for biological applications.

A single-shot water-immersion digital holographic microscope combined with broadband (white light) illumination mode is presented. This double imaging platform allows conventional incoherent visualization with phase holographic imaging of inspected samples. The holographic architecture is implemented at the image space (that is, after passing the microscope lens), thus reducing the sensitivity of the system to vibrations and/or thermal changes in comparison to regular interferometers. Because of the off-axis holographic recording principle, quantitative phase images of live biosamples can be recorded in a single camera snapshot at full-field geometry without any moving parts. And, the use of water-immersion imaging lenses maximizes the achievable resolution limit. This dual-mode microscope platform is first calibrated using microbeads, then applied to the characterization of fixed cells (neuroblastoma, breast cancer, and hippocampal neuronal cells) and, finally, validated for visualization of dynamic living cells (hippocampal neurons).

Superresolved spatially multiplexed interferometric microscopy.

Superresolution capability by angular and time multiplexing is implemented onto a regular microscope. The technique, named superresolved spatially multiplexed interferometric microscopy (S2MIM), follows our previously reported SMIM technique [Opt. Express22, 14929 (2014)OPEXFF1094-408710.1364/OE.22.014929, J. Biomed. Opt.21, 106007 (2016)JBOPFO1083-366810.1117/1.JBO.21.10.106007] improved with superresolved imaging. All together, S2MIM updates a commercially available non-holographic microscope into a superresolved holographic one. Validation is presented for an Olympus BX-60 upright microscope with resolution test targets.

An alternative clinical routine for subjective refraction based on power vectors with trial frames.

PURPOSE: Subjective refraction determines the final point of refractive error assessment in most clinical environments and its foundations have remained unchanged for decades. The purpose of this paper is to compare the results obtained when monocular subjective refraction is assessed in trial frames by a new clinical procedure based on a pure power vector interpretation with conventional clinical refraction procedures. METHODS: An alternative clinical routine is described that uses power vector interpretation with implementation in trial frames. Refractive error is determined in terms of: (i) the spherical equivalent (M component), and (ii) a pair of Jackson Crossed Cylinder lenses oriented at 0°/90° (J0 component) and 45°/135° (J45 component) for determination of astigmatism. This vector subjective refraction result (VR) is compared separately for right and left eyes of 25 subjects (mean age, 35 ± 4 years) against conventional sphero-cylindrical subjective refraction (RX) using a phoropter. The VR procedure was applied with both conventional tumbling E optotypes (VR1) and modified optotypes with oblique orientation (VR2). RESULTS: Bland-Altman plots and intra-class correlation coefficient showed good agreement between VR, and RX (with coefficient values above 0.82) and anova showed no significant differences in any of the power vector components between RX and VR. VR1 and VR2 procedure results were similar (p ≥ 0.77). CONCLUSIONS: The proposed routine determines the three components of refractive error in power vector notation [M, J0 , J45 ], with a refraction time similar to the one used in conventional subjective procedures. The proposed routine could be helpful for inexperienced clinicians and for experienced clinicians in those cases where it is difficult to get a valid starting point for conventional RX (irregular corneas, media opacities, etc.) and for refractive situations/places with inadequate refractive facilities/equipment.